Methods for reprogramming cells and uses thereof

ABSTRACT

An in vitro human cardiac multi potent or unipotent cell that has the ability to proliferate; may be maintained in standard cardiac stem cell media; can differentiate to a progenitor, precursor, or somatic cell; has the characteristics of a cardiac stem cell, a cardiac precursor cell, or a cardiac progenitor cell; does not exhibit uncontrolled growth, teratoma formation, or tumor formation in vivo; expresses one or more markers of a multipotent, unipotent or somatic cell not characteristic of a cardiac stem cell, a cardiac precursor cell, or a cardiac progenitor cell; and is derived from the reprogramming of a somatic cell, a progenitor cell or a stem cell that exhibits at least a transient increase in intracellular levels of at least one reprogramming agent; wherein the cell comprises at least one transiently expressed polypeptide or an expression vector.

FIELD OF THE INVENTION

The present invention relates to the field of eukaryotic cellreprogramming, and particularly to cell dedifferentiation. The inventionis also concerned with methods of generating stable Neural Stem-LikeCells (NSLCs) from human somatic cells (and other cells) and the use ofthe cells so generated in human therapy.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. §1.52(e). Thename of the ASCII text file for the Sequence Listing is 24258038_1.TXT,the date of creation of the ASCII text file is Sep. 20, 2016, and thesize of the ASCII text file is 1.50 KB.

BACKGROUND OF THE INVENTION Cell Reprogramming

There is a desire in the medical, scientific, and diagnostic fields toreprogram an easily obtainable cell into a cell that is generally harderto obtain, or to reprogram a cell to have new or differentfunctionalities, without fusing or exchanging material with an oocyte oranother stem cell.

According to a first mechanism, a stem cell can naturally divide ordifferentiate into another stem cell, progenitor, precursor, or somaticcell. According to a second mechanism, somatic cell can sometimestransiently change its phenotype or express certain markers when placedin certain conditions, and then revert back when placed back into theoriginal conditions. According to a second mechanism, the phenotype ofmany cells can be changed through forced expression of certain genes(for example, stably transfecting the c-myc gene into fibroblasts turnsthem into immortal cells having neuroprogenitor characteristics),however once this forced gene expression is removed, the cells slowlyrevert back to their original state. Therefore, none of the three abovemechanisms should be considered true reprogramming: the first isconsidered natural differentiation which is part of a cell program thatis already in place (going from a more undifferentiated to a moredifferentiated state), the second is a transient phenotypical change,and the third is a constantly forced cell type. A true stem cell: (i)self-renews almost ‘indefinitely’ (for significantly longer than asomatic cell), (ii) is not a cancerous cell, (iii) is not artificiallymaintained by forced gene expression or similar means (must also be ableto be maintained in standard stem cell media), (iv) can differentiate toprogenitor, precursor, somatic or other more differentiated cell type(of the same lineage), and (v) has all the characteristics of a stemcell and not just certain markers or gene expression or morphologicalappearance.

Despite the numerous scientific and patent publications claimingsuccessful reprogramming or dedifferentiation, generally into a stemcell, almost all of these publications do not disclose truereprogramming because they fall under one of the mechanisms mentionedabove. For instance, Bhasin (WO2010/088735), Cifarelli et al.(US2010/0003223), Kremer et al. (US2004/0009595), and Winnier et al.(US2010/0047908) all refer to reprogramming, dedifferentiation, and/orobtained stem cells (or progenitors) as phenotypical cell changes basedonly on a change in cell surface markers after culture in differentmedia with supplements, with no evidence of true reprogramming or anactual stem cell (non-cancerous self-renewal with stem cells markers andno differentiation markers). The same is true for Benneti(WO2009/079007) who used increased expression of Oct4 and Sox2. Others,such as Akamatsu et al. (WO2010/052904) and You et al. (WO2007/097494,US2009/0246870), refer to having made stem cells, but these came aboutthrough constant artificial gene induction delivered by retrovirus(similar to cMyc) with no evidence of true stem cells that are notimmortal/tumorigenic, and stable instead of transient. Others, such asChen et al. (US2005/0176707) and You et al. (US2009/0227023), have made“multipotent cells”, but not stem cells. In addition these alledgedmultipotent cells were not stable (in the case of You et al. the cellscould not even proliferate) and both used constant media supplements andconditions to force the phenotypical change. Yet others, such as Oliveriet al. (WO2009/018832) and Zahner et al. (US2002/0136709), have claimedthe making of pluripotent, totipotent, multipotent, and/or unipotentcells automatically through genome-wide DNA demethylation and histoneacetylation, but with no evidence of a stable, non-cancerous, true cellline.

True reprogramming appears to have been achieved with inducedpluripotent stem cells (iPS cells) created independently by Yamanaka'sgroup (Takahashi et al., 2007) and Thomson's group (Yu et al., 2007),and potentially by others before them, and although many of these cellswere later found to be cancerous, some of them were not. These cells canbe induced by true reprogramming since it was later shown that they canalso be induced by non-gene integrating transient transfection (Soldneret al., 2009; Woltjen et al., 2009; Yu et al., 2009) as well as by RNA(Warren et al., 2010) or protein (Kim et al., 2009; Zhou et al., 2009)alone or by small molecules (Lyssiotis et al., 2009), and by similarmethods. However, these cells are essentially identical to embryonicstem cells and have the same problems of uncontrolled growth, teratomaformation, and potential tumor formation.

A more desirable option is to have multipotent stem cells orpluripotent-like cells whose lineage and differentiation potential ismore restricted so that they do not readily form teratomas anduncontrolled growth. There is thus a need for methods of creatingmultipotent stem cells, multipotent stem-like cells, and stem-like cellsand method of reprogramming or transforming easily obtainable cells tohighly desirable multipotent stem cells, multipotent stem-like cells,and stem-like cells.

Neural Stem-Like Cells (NSLC)

Repairing the central nervous system (CNS) is one of the frontiers thatmedical science has yet to conquer. Conditions such as Alzheimer'sdisease, Parkinson's disease, and stroke can have devastatingconsequences for those who are afflicted. A central hope for theseconditions is to develop cell populations that can reconstitute theneural network, and bring the functions of the nervous system back inline. For this reason, there is a great deal of evolving interest inneural stem and progenitor cells. Up until the present time, it wasgenerally thought that multipotent neural progenitor cells commit earlyin the differentiation pathway to either neural restricted cells or gliarestricted cells.

Neural stem cells have promise for tissue regeneration from disease orinjury; however, such therapies will require precise control over cellfunction to create the necessary cell types. There is not yet a completeunderstanding of the mechanisms that regulate cell proliferation anddifferentiation, and it is thus difficult to fully explore theplasticity of neural stem cell population derived from any given regionof the brain or developing fetus.

The CNS, traditionally believed to have limited regenerativecapabilities, retains a limited number of neural stem cells inadulthood, particularly in the dentate gyrus of the hippocampus and thesubventricular zone that replenishes olfactory bulb neurons (Singec I etal., 2007; Zielton R, 2008). The availability of precursor cells is akey prerequisite for a transplant-based repair of defects in the maturenervous system. Thus, donor cells for neural transplants are largelyderived from the fetal brain. This creates enormous ethical problems, inaddition to immuno-rejection, and it is questionable whether such anapproach can be used for the treatment of a large number of patientssince neural stem cells can lose some of their potency with each celldivision.

Neural stem cells provide promising therapeutic potential forcell-replacement therapies in neurodegenerative disease (Mimeault etal., 2007). To date, numerous therapeutic transplantations have beenperformed exploiting various types of human fetal tissue as the sourceof donor material. However, ethical and practical considerations andtheir inaccessibility limit the availability as a cell source fortransplantation therapies (Ninomiy M et al., 2006).

To overcome barriers and limitations to the derivation of patientspecific cells, one approach has been to use skin cells and inducing thetrans-differentiation to neural stem cells and/or to neurons (Levesqueet al., 2000). Transdifferentiation has been receiving increasingattention during the past years, and trans-differentiation of mammaliancells has been achieved in co-culture or by manipulation of cell cultureconditions. Alteration of cell fate can be induced artificially in vitroby treatment of cell cultures with microfilament inhibitors (Shea etal., 1990), hormones (Yeomans et al., 1976), and Calcium-ionophores(Shea, 1990; Sato et al., 1991). Mammalian epithelial cells can beinduced to acquire muscle-like shape and function (Paterson and Rudland,1985), pancreatic exocrine duct cells can acquire an insulin-secretingendocrine phenotype (Bouwens, 1998a, b), and bone marrow stem cells canbe differentiated into liver cells (Theise et al., 2000) and intoneuronal cells (Woodbury et al., 2000). Other such as Page et al. (US2003/0059939) have transdifferentiated somatic cells to neuronal cellsby culturing somatic cells in the presence of cytoskeletal, acetylation,and methylation inhibitors, but after withdrawal of the priming agent,neuron morphology and established synapses last for not much than a fewweeks in vitro, and complete conversion to a fully functional and stabletype of neuron has never been demonstrated. These are thus transientcell phenotypes. Complete conversion to a fully functional and stabletype of neuroprogenitor or neural stem cell has also never beendemonstrated. Acquisition of a stable phenotype followingtransdifferentiation has been one of the major challenges facing thefield.

Thus, there is a need in the biomedical field for stable, potent, andpreferably autologouos neural stem cells, neural progenitor cells, aswell as neurons and glial cells for use in the treatment of variousneurological disorders and diseases. The same is true for many othertypes of cells. Recently, evidence have been obtained that genes of thebasic Helix-Loop-Helix (bHLH) class are important regulators of severalsteps in neural lineage development, and over-expression of severalneurogenic bHLH factors results in conversion of non-determined ectoderminto neuronal tissue. Proneural bHLH proteins control thedifferentiation into progenitor cells and their progression through theneurogenic program throughout the nervous system (Bertrand et al.,2002). MASH1, NeuroD, NeuroD2, MATH1-3, and Neurogenin 1-3 are bHLHtranscription factors expressed during mammalian neuronal determinationand differentiation (Johnson et al., 1990; Takebyashi et al., 1997;McCormick et al., 1996; Akazawa et al., 1995). Targeted disruptions ofMASH1, Ngn1, Ngn2 or NeuroD in mice lead to the loss of specific subsetsof neurons (Guillemot et al., 1993; Fode et al., 1998; Miyata et al.,1999).

U.S. Pat. No. 6,087,168 (Levesque et al.,) describes a method forconverting or transdifferentiating epidermal basal cells into viableneurons. In one example, this method comprises the transfection of theepidermal cells with one or more expression vector(s) containing atleast one cDNA encoding for a neurogenic transcription factorresponsible for neural differentiation. Suitable cDNAs include:basic-helix-loop-helix activators, such as NeuroD1, NeuroD2, ASH1, andzinc-finger type activators, such as Zic3, and MyT1. The transfectionstep was followed by adding at least one antisense oligonucleotide knownto suppress neuronal differentiation to the growth medium, such as thehuman MSX1 gene and/or the human HES1 gene (or non-human, homologouscounterparts). Finally, the transfected cells were grown in the presenceof a retinoid and a least one neurotrophin or cytokine, such as brainderived neurotrophic factor (BDNF), nerve growth factor (NGF),neurotrophin 3 (NT-3), or neurotrophin 4 (NT-4). This technology yields26% of neuronal cells; however, neither functionality nor stability ofthese cells was established. In addition, neural stem cells orneuroprogenitor cells are not produced according to this method.

A later process (Levesque et al., 2005; U.S. Pat. No. 6,949,380)mentions the conversion of the epidermal basal cell into a neuralprogenitor, neuronal, or glial cell by exposing the epidermal basal cellto an antagonist of bone morphogenetic protein (BMP) and growing thecell in the presence of at least one antisense oligonucleotidecomprising a segment of a MSX 1 gene and/or HES1 gene. However, there isno evidence or examples that any neural progenitors or glial cells wereproduced according to this method, let alone any details or evidencethat morphological, physiological or immunological features of neuronalcells was achieved. In addition, since there is also no information onfunctionality, stability, expansion, and yield about the cells which mayor may not have been produced, it is possible that these cells actuallyare skin-derived precursor cells (Fernandes et al., 2004) that have beendifferentiated into neuronal cells.

In view of the above, there is thus a need for stable, potent, andpreferably autologouos neural stem cells, neural progenitor cells,neurons and glial cells, as well as other types of cells, stem cells andprogenitor cells. There is also a need for methods that could result intrue cell dedifferentiation and cell reprogramming.

The present invention addresses these needs and provides various typesof stem-like and progenitor-like cells and cells derived ordifferentiated from these stem-like or progenitor-like cells, as well asmethods that can result in true cell dedifferentiation and cellreprogramming.

Additional features of the invention will be apparent from a review ofthe disclosure, figures and description of the invention herein.

SUMMARY OF THE INVENTION

The present invention relates to stem-like and progenitor-like cells andcells derived or differentiated from these stem-like or progenitor-likecells. The invention further relates to methods for celldedifferentiation and cell reprogramming. The invention further featurescompositions and methods that are useful for reprogramming cells andrelated therapeutic compositions and methods.

One particular aspect relates to the development of a technology toreprogram a somatic cell or non-neuronal cell to a cell having one ormore morphological physiological, and/or immunological features of aneural stem cell and which possess the capacity to differentiate alongneuronal and glial lineages. According to some embodiments, theinvention is more particularly concerned with methods of generatingstable Neural Stem-Like Cells (NSLCs) from human somatic cells, humanprogenitor cells and/or of human stem cells, as well as cells, celllines and tissues obtained by using such methods.

The invention further relates to compositions and methods to inducede-differentiation of human somatic cells into Neural Stem-Like Cellsthat express neural stem cell specific markers. According to the presentinvention it is possible to effect the conversion of cells to varioustypes of differentiated neuronal cells that can be created from a singlecell type taken from an individual donor and then reprogrammed andtransplanted into the same individual. Upon induction cells according tothe invention express neural stem-cell specific markers and becomeNeural Stem-Like cells.

According to one particular aspect, the invention relates to a method oftransforming a cell of a first type to a desired cell of a differenttype. The comprises i) obtaining a cell of a first type; ii) transientlyincreasing in the cell of a first type intracellular levels of at leastone reprogramming agent, whereby the transient increase induces director indirect endogenous expression of at least one gene regulator; iii)placing the cell in conditions for supporting the growth and/or thetransformation of the desired cell and maintaining intracellular levelsof the at least one reprogramming agent for a sufficient period of timeto allow stable expression of the at least one gene regulator in absenceof the reprogramming agent; and iv) maintaining the cell in cultureconditions supporting the growth and/or the transformation of thedesired cell. Such conditions are maintained for a sufficient period oftime to allow a stable expression of a plurality of secondary genes.According to the invention the expression of one or more of thesecondary genes is characteristic of phenotypical and functionalproperties of the desired cell while being not characteristic ofphenotypical and functional properties of an embryonic stem cell.Therefore, at the end of the period of time, the desired cell of adifferent type is obtained.

According to another particular aspect, the invention relates to amethod of transforming a cell of a first type to a cell of a seconddifferent type. The method comprises contacting the cell of a first typewith one or more agents capable of increasing within said cell levels ofat least one reprogramming agent and directly or indirectly remodelingthe chromatin and/or DNA of the cell. The at least one reprogrammingagent is selected for inducing directly or indirectly the expression ofmorphological and functional characteristics of a desired cell of adifferent type or different cell lineage.

According to another aspect, the invention relates to a method oftransforming a cell of a first type to a cell of a second differenttype. The method comprises contacting the chromatin and/or DNA of a cellof a first type with an agent capable of remodeling chromatin and/or DNAof said cell; and increasing intracellular levels of at least onereprogramming agent. The at least one reprogramming agent is selectedfor inducing directly or indirectly the expression of morphological andfunctional characteristics of a desired cell of a different type or celllineage.

A further aspect of the invention relates to a method of transforming acell of a first type to a cell of a desired cell of a different type,comprising increasing intracellular levels of at least one reprogrammingagent, wherein the at least one reprogramming agent is selected forinducing directly or indirectly the expression of morphological andfunctional characteristics of a desired second cell type; andmaintaining the cell of a first type in culture conditions forsupporting the transformation of the desired cell for a sufficientperiod of time to allow stable expression of a plurality of secondarygenes whose expression is characteristic of phenotypical and functionalproperties of the desired cell, wherein at least one of the secondarygenes is not characteristic of phenotypical and functional properties ofan embryonic stem cell. At the end of the period of time the desiredcell of a different type is obtained and the obtained cell is furthercharacterized by a stable repression of a plurality of genes expressedin the first cell type.

A further aspect of the invention concerns a process wherein a cell of afirst type is reprogrammed to a desired cell of a different type, theprocess comprising:

-   -   a transient increase of intracellular levels of at least one        reprogramming agent, wherein the at least one reprogramming        agent induces a direct or indirect endogenous expression of at        least one gene regulator, and wherein the endogenous expression        of the said at least one gene regulator is necessary for the        existence of the desired cell of a different type;    -   a stable expression of said at least one gene regulator;    -   stable expression of a plurality of secondary genes, wherein the        stable expression of the secondary genes is the result of the        stable expression of the at least one gene regulator, and        wherein: (i) stable expression of the plurality of secondary        genes is characteristic of phenotypical and/or functional        properties of the desired cell, (ii) stable expression of at        least one of said secondary genes is not characteristic of        phenotypical and functional properties of an embryonic stem        cell, and wherein (i) and (ii) are indicative of successful        reprogramming of the cell of the first type to the desired cell        of the different type.

In particular embodiments, the at least one reprogramming agent in theprocess is a Msi1 polypeptide, or a Ngn2 polypeptide together with aMDB2 polypeptide. In particular embodiments, the at least one generegulator is Sox2 Msi1, or both. In additional embodiments the at leastone gene regulator may is one or more of the genes listed in Table A forNeural Stem-Like Cells.

According to another aspect, the invention relates to a method ofobtaining a Stem-Like Cell (SLC), comprising:

-   -   i) providing a cell of a first type;    -   ii) transiently increasing in the cell intracellular levels of        at least one reprogramming agent, whereby the transient increase        induces direct or indirect endogenous expression of at least one        gene regulator;    -   iii) placing the cell in conditions for supporting the        transformation into the stem-like cell and maintaining        intracellular levels of the at least one reprogramming agent for        a sufficient period of time to allow stable expression of the at        least one gene regulator in absence of the reprogramming agent;    -   iv) maintaining the cell in culture conditions for supporting        the transformation into the stem-like cell for a sufficient        period of time to allow stable expression of a plurality of        secondary genes whose expression is characteristic of        phenotypical and/or functional properties of the stem-like cell,        wherein at least one of the secondary genes is not        characteristic of phenotypical and functional properties of an        embryonic stem cell. At the end of said period of time a        stem-like cell is obtained.

According to another aspect, the invention relates to a method ofobtaining a Stem-Like Cell. The method comprises increasingintracellular levels of at least one polypeptide specific to the desiredstem cell type that is able to drive directly or indirectlytransformation of the cell of the first type into the Stem-Like Cell.For increasing the yield or type of Stem-Like Cell, the method mayfurther comprises contacting chromatin and/or DNA of a cell of a firsttype with a histone acetylator, an inhibitor of histone deacetylation, aDNA demethylator, and/or an inhibitor of DNA methylation; and/orincreasing intracellular levels of at least one other polypeptidespecific to the desired stem cell type that is able to drive directly orindirectly transformation of the cell of the first type into a Stem-LikeCell.

According to another aspect, the invention relates to a method ofobtaining a Neural Stem-Like Cell (NSLC). The method comprisesincreasing intracellular levels of at least one neural stem cellspecific polypeptide that is able to drive directly or indirectlytransformation of the cell of the first type into a NSLC. For increasingthe yield or type of NSLC, the method further comprises. contactingchromatin and/or DNA of a cell of a first type with a histoneacetylator, an inhibitor of histone deacetylation, a DNA demethylator,and/or an inhibitor of DNA methylation; and/or increasing intracellularlevels of at least one other neural stem cell specific polypeptide thatis able to drive directly or indirectly transformation of the cell ofthe first type into a NSLC.

Another aspect of the invention concerns a method of obtaining a NeuralStem-Like Cell (NSLC). In one embodiment the method comprisestransfecting a skin cell with a polynucleotide encoding Musashi1,Musashi1 and Neurogenin 2, Musashi1 and Methyl-CpG Binding DomainProtein 2 (MBD2), or Neurogenin 2 and Methyl-CpG Binding Domain Protein2, thereby reprogramming the skin cell into a NSLC. In anotherembodiment the method comprises exposing a skin cell to: (i) aninhibitor of histone deacetylation, (ii) an inhibitor of DNAmethylation, (iii) a histone acetylator, and/or (iv) a DNA demethylatorsuch as a MBD2 polypeptide and/or transfecting with a polynucleotideencoding a MBD2 polypeptide; and further transfecting the cell (eithersimultaneously, before, or afterwards) with a polynucleotide encodingMUSASHI1 and/or with a polynucleotide encoding NGN2, therebyreprogramming the skin cell into a NSLC. Some other cells, such askeratinocytes and CD34⁺ cells, can also be used and reprogrammed.

In one particular embodiment, the method of obtaining a Neural Stem-LikeCell (NSLC), comprises:

-   -   providing a cell of a first type;    -   introducing into the cell one or more polynucleotide capable of        transient expression of one or more the following polypeptides:        Musashi1 (Msi1); a Musashi1 (Msi1) and a Neurogenin 2 (Ngn2); a        Musashi1 (Msi1) and methyl-CpG binding domain protein 2 (MBD2);        and Neurogenin 2 (Ngn2) and methyl-CpG binding domain protein 2        (MBD2); and    -   placing the cell in culture conditions supporting the        transformation into a NSLC for a sufficient period of time to        allow a stable expression of a plurality of genes whose        expression is characteristic of phenotypical and functional        properties of a NSLC.

At the end of the period of time a NSLC is obtained and the obtainedNSLC is further characterized by a stable repression of a plurality ofgenes expressed in the first cell type.

According to another embodiment, the method of obtaining a NeuralStem-Like Cell (NSLC), comprises:

-   -   providing a cell of a first type which is not a NSLC;    -   increasing intracellular levels of at least one neural stem cell        specific polypeptide, wherein the polypeptide is capable of        driving directly or indirectly transformation of the cell of the        first type into a NSLC; and    -   contacting the chromatin and/or DNA of the cell of a first type        with a histone acetylator, an inhibitor of histone        deacetylation, a DNA demethylator, and/or a chemical inhibitor        of DNA methylation.

According to another embodiment, the method of obtaining a NeuralStem-Like Cell (NSLC), comprises:

-   -   obtaining a non-NSLC;    -   co-transfecting the non-NSLC with a first polynucleotide        encoding a MBD2 polypeptide and with at least one second        polynucleotide encoding a MUSASHI1 polypeptide and/or encoding a        NGN2 polypeptide;    -   placing the co-transfected cell in culture conditions for        supporting the transformation of NSLC until said NSLC is        obtained.

Certain aspects of the invention concerns isolated cells, cell lines,compositions, 3D assembly of cells, and tissues comprising cellsobtained using the methods described herein. Additional aspects concernsthe use of such isolated cells, cell lines, compositions, 3D assembly ofcells, and tissues of medical treatment and methods of regenerating amammalian tissue or organ.

Yet, a further aspect concerns a method for repairing or regenerating atissue in a subject. In one embodiment the method comprises theadministration of a reprogrammed cell as defined herein to a subject inneed thereof, wherein the administration provides a dose of reprogrammedcells sufficient to increase or support a biological function of a giventissue or organ, thereby ameliorating the subject's condition.

The benefits of the present invention are significant and include lowercost of cell therapy by eliminating the need of immuno-suppressiveagents, no need for embryos or fetal tissue, thus eliminating ethicaland time constraints, lower cost of production, and no health risks dueto possible transmission of viruses or other disease. In addition, sincethe cells are created fresh, they tend to be more potent than cells thathave been passaged multiple times.

Additional aspects, advantages and features of the present inventionwill become more apparent upon reading of the following non-restrictivedescription of preferred embodiments which are exemplary and should notbe interpreted as limiting the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a panel of light micrograph (10×) presenting cell morphologychanges of untransfected and transfected cells with Msi1 and MBD2 atvarious time points.

FIG. 2 is a panel of photomicrographs obtained using Cellomics™ (10×)and revealing NCAM positive cells in transfected cells with Msi1 or Ngn2in the presence of MBD2. HFFs were pre-treated with cytochalasin B (10μg/ml) and transfected with pCMV6-XL5-Msi1 and pCMV6-XL5-MBD2 orpCMV6-XL4-Ngn2 and pCMV6-XL5-MBD2. After 24 h following transfection,the medium was changed and cells were cultured in proliferation medium(NPBM, Lonza) supplemented with EGF (20 ng/ml) and bFGF (20 ng/ml) forone week. Differentiation was induced by changing the medium to NbActive(BrainBits™) supplemented with NGF (20 ng/ml), bFGF (20 ng/ml), ATRA(50) and Forskolin (10 μM). Cells were incubated at 37° C., 5% CO₂, 5%O₂ for 20 days.

FIG. 3 is a panel of photomicrographs obtained using Cellomics™ (10×)and revealing MAP2b positive cells in transfected cells with Msi1 orNgn2 in the presence of MBD2. MAP2b positive cells were undetectable inuntransfected cells and cells transfected with Pax6/MBD2. HFFs werepre-treated with cytochalasin B (10 μg/ml) and transfected withpCMV6-XL5-Msi1, pCMV6-XL4-Ngn2 or pCMV6-XL5-Pax6, and pCMV6-XL5-MBD2.After 24 h following transfection, the medium was changed and cells werecultured in proliferation medium (NPBM, Lonza) supplemented with EGF (20ng/ml. Peprotech) and bFGF (20 ng/ml, Peprotech) for one week.Differentiation was induced by changing the medium to NbActive(BrainBits™) supplemented with NT-3 (20 ng/ml), bFGF (20 ng/ml), ATRA(50) and Forskolin (10 μM). Cells were incubated at 37° C., 5% CO₂, 5%O₂ for 2 weeks.

FIG. 4. (A) is a panel of photographs showing that neurospheres formedby NSLCs from Example V were completely dissociated into single cellsuspensions using Accutase and one single cell was monitored over timeto reveal neurosphere formation capacity (Light microscope observation).Neurospheres stained positive for Sox2. (B) is a panel of photographsfrom immunohistochemistry results obtained using Cellomics™.Immunohistochemistry was performed, on day 20, to detect makers forneurospheres and compared to expression levels in neurospheres formed bynormal human neuroprogenitor cells (hNPC, Lonza). In addition to Sox2,cells stained positive for the neural stem cells markers Musashi, CD133,Nestin, and GFAP. Cells also stained positive for βIII-tubulin (a markerfor neurons), O4 (a marker for oligodendrocytes), and GFAP (a marker forastrocytes), indicating the tri-potent differentiation potential of bothsets of cells (NSLC and hNPC), and negative for NGFrec and NeuN (markersfor differentiated neurons) indicating that the cells were notterminally differentiated.

FIG. 5 is a panel photomicrographs from immunohistochemistry resultsobtained using Cellomics™. Immunohistochemistry was performed on HFFs,NSLCs, and hNPCs to detect expression of markers for fibroblasts as wellas neural stem cells (Sox2, Nestin, GFAP) in adherent cultures (thatprevented cells from floating and forming neurospheres). Nuclei werestained with Hoechst (upper level pictures). HFFs expressed fibroblastsmarkers while NSLCs created from these HFFs did not. In comparison, theNSLCs expressed neural stem cell markers similarly to hNPCs while theHFFs did not express any of these markers.

FIG. 6 is a panel photomicrographs showing Human NSLCs. Human NSLCs wereinduced to differentiate into neuronal lineages in the presence of NS-Adifferentiation medium (StemCell Technologies) in the presence of BDNF(20 ng/ml, Peprotech) and bFGF (40 ng/ml, Peprotech) for three weeks. Atdifferent time point of differentiation, immunostaining using Cellomics™(10×) revealed differentiation of the cells as shown by the decrease ofSox2 positive cells and increase in the number and intensity of stainingof p75, βIII-tubulin and GABA positive cells, as well as differentiatedmorphology, while the total number of cells increased as shown byHoechst staining.

FIG. 7 is another panel of photomicrographs. HFF, Keratinocytes, andCD34+ were transfected with pCMV6-Msi1-Ngn2 and pCMV6-XL5-MBD2. After 24h following transfection, the medium was changed to proliferation medium(StemCell Technologies) supplemented with EGF (20 ng/ml. Peprotech) andbFGF (20 ng/ml, Peprotech) for two week and then analyzed.Photomicrographs using Cellomics™ (10×) show that NSLCs created from allthree types of cells are positive for Nestin, Sox2 and GFAP (markers forneural stem cells), while the original HFFs are not.

FIG. 8 is panel of photomicrographs showing the effect of CDM medium onthe trans-differentiation of HFF towards neurons. HFF were pre-treatedwith cytochalasin B (10 μg/ml) and histone deacetylation inhibitor (VPA,4 mM) and DNA methylation inhibitor (5-Aza, 5 μM and cultured in CDMmedium containing 3:1 ratio of Dulbecco's modified Eagle medium (DMEM,high glucose (4.5 g/L) with L-glutamine and sodium pyruvate) and Ham'sF-12 medium supplemented with the following components: EGF(4.2×10⁻¹⁰M), bFGF (2.8×10⁻¹⁰M), ITS (8.6×10⁻⁵M), dexamethasone(1.0×10⁻⁷M), L-ascorbic acid phosphate magnesium salt n-hydrate(3.2×10⁻⁴M), L-3,3′,5-triiodothyronine (2.0×10⁻¹⁰M), ethanolamine(10⁻⁴M), GlutaMAX™ (4×10³M), glutathione (3.3×10⁻⁶M). After 24 h theculture medium was replaced with 75% of CDM medium and 25% of NeuronalProliferation medium (Lonza, Cat#CC-3210); during the following 3 days,the ratio of the medias were changed to 50%:50%, 25%:75%, and then 100%Neuronal Proliferation medium by the third day. Photomicrographs weretaken by Cellomics™ (10×) after immunostaining the cells withβIII-tubulin (neuronal marker) and Hoechst (to stain nuclei) atdifferent time-points. Cells started trans-differentiating within a fewdays and the trans-differentiated cells were βIII-tubulin positive;however after one week a spontaneous reversion to fibroblastic shape andloss of βIII-tubulin expression was observed.

FIG. 9 is panel of photomicrographs showing characterization ofreprogrammed cells within CDM at different time points following thetransfection with Msi1 and Ngn2. The transfected cells were treated withCytochalasin B (10 μg/ml), VPA (4 mM) and 5-AZA (5 μM) resulting in adisruption of the microfilaments and rounding up of the cells andloosening of the chromatin. Immunohistochemistry on the 3-DimensionalCDM was performed after one and two weeks using Cellomics™ (10×). Thecells were positive for neuronal mature marker, such as MAP2b, but wereabsent in the untransfected control CDM.

FIG. 10 is another panel of photomicrographs. Cells within Day 4 CDMwere lipotransfected with the two vectors pCMV6-XL5-Msi1 andpCMV6-XL4-Ngn2 individually or together in combination withpCMV-XL5-MBD2 for a period of 6 hours. In parallel, transfection wasperformed on fresh HFFs after the 6 hours using Nucleofection, and thesefresh HFFs were placed on top of the CDM at the same time as thelipofectamine media was changed to fresh CDM medium after 6 hours. After24 hours the medium was changed to Neural proliferation medium (NPBM,Lonza) with the presence of Noggin (50 ng/ml, Peprotech), recombinanthFGF (20 ng/ml, Peprotech), and recombinant hEGF (20 ng/ml, Peprotech)for one week. Differentiation was induced at day 7, by adding NS-Adifferentiation medium (StemCell Technologies) for 24 days.Immunohistochemistry was performed at various time points usingCellomics™ (10×). The CDM was stained with a specific antibody againstNestin (a marker for neural stem cells), and cells within the CDMexpressed Nestin at all timepoints tested (Day 8, 15, and 21) followingtransfection. Cells within the untransfected control CDM did not expressany Nestin.

FIG. 11 is a panel showing a picture of a polyacrylamide gelelectrophoresis. NSLCs grown as adherent cultures or suspension cultures(as neurospheres) both express telomerase (which is expresses in allstem cells, but not in normal differentiated somatic cells). Both early(p5) and late (p27) passage NSLCs express telomerase. (The original HFFsfrom which the NSLCs were created did not express telomerase.) Thesamples (NSLCs) were spun down and protein concentration of thesupernatant was determined using the BCA Assay. 900 ng of protein fromeach cell extract was added directly to the TRAP reaction mixturecontaining TRAP reaction buffer, dNTPs, template substrate (TS) primer,TRAP primer mix and Taq polymerase. The reaction mixtures were incubatedat 30° C. for 30 minutes for template synthesis, followed by a PCRprocedure (95° C./15 min for initial denaturation, 94° C./30 sec, 59°C./30 sec, 72° C./1 min for 32 cycles) for amplification of the extendedtelomerase products. To detect telomerase activity, polyacrylamide gelelectrophoresis (PAGE) was performed for the reaction products on a 10%non-denaturing TBE gel. After electrophoresis, the gel was stained withSYBR® Green I Nucleic Acid Gel Stain for 30 minutes, followed by imagecapture using the Gel-Documentation System (Alpha Innotech). All 4samples were telomerase positive (as indicated by the TRAP productladder).

FIG. 12 is a panel showing a picture showing Southern blot analysis oftwo different NSLC samples analyzed for Msi1 and Ngn2 gene integrationtwo weeks after transient transfection. The Dig-labeled PCR proberevealed distinct signals in the positive control samples where theMsi1/Ngn2 plasmid DNA was spiked into HFF genomic DNA for theequivalence of 1, 10 or 100 integrations per genome. There were a fewweak and identical bands that appeared in the restriction enzymedigested genomic DNA from untransfected HFF and NSLC samples #1 and #2,suggesting that there was no plasmid DNA integration into the genomicDNA of NSLCs. These faint bands may represent the endogenous Ngn2 genesince the 1.2 kb Dig-labeled PCR probe contains a small part of the Ngn2gene. There were positive signals in the lane of the DNA kb ladder asthe bands belong to a number of plasmids digested to completion withappropriate restriction enzymes (NEB). This data shows that no, or onlya tiny number of, NSLCs had plasmid integration into the host genomeafter transient transfection, and that the transiently transfected geneswere only present in the cells for a short period of time (less than twoweeks).

FIG. 13 is a panel with a line graph and a bar graph showing improvementand significantly better clinical scores in EAE mice treated with NSLCs.Female 8 weeks old C57BL/6 mice were immunized with MOG₃₅₋₅₅ (SheldonBiotechnology Centre McGill University) in CFA containing 5 mg/ml ofdesiccated (killed and dried) Mycobacterium tuberculosis H37Ra (Difco,inc) at two sites on the back, and injected with 200 ng of pertussistoxin (List Biological Laboratories, Inc) in PBS intraperitoneally ondays 0 and 2. Once the mice started showing symptoms of EAE (on Day 13post-immunization), they were intravenously injected with 200 μl of NSLC(1 million cells), hNPC (1 million cells), saline, or saline withcyclosporine. All mice except the saline control group received dailyinjections of cyclosporine. Mice were scored daily for clinical disease;data represent average daily scores. Mice that received a singleinjection of NSLCs had a significantly lower disease severity than micethat received hNPCs or cyclosporine alone.

FIG. 14 is a line graph showing the results of rotarod assessmentsaccording to Example XVII part 2. Rats were trained on the rotarod priorto the start of the experiment. Rats were placed on a stationary androtating rotarod (rotating at 20 rpm) and the amount of time spent bythe rats walking on the rotarod before falling off was monitored.Measurements were taken before (pre-surgery) and after (post-surgery)surgical left brain hemisphere ablation and treatment. The data pointsrepresent the mean number of falls by each animal during each 60 secondtesting session carried out at a constant speed of 20 rpm. Each groupconsisted of eight rats.

FIG. 15 is a line graph showing the results of the walking beamassessments according to Example XVII part 2. Rats were measured ontheir ability to cross a 100 cm long beam after surgical left brainhemisphere ablation and treatment. Two days after surgery, all groupsfail to pass the test, and the animals are not able to stay in balanceon the beam. One week after the surgery, all the animals show animprovement on their walking capacity, but no significant difference wasnoticeable between the different treated groups. From week 4 until week26, the animals treated with NSLCs show significant improvement in theirwalking capacity compared to the other groups.

FIG. 16 is a panel showing photographs of ADSCs transiently transfectedwith various pluripotent vectors using nucleofector as described inExample XIX. Following the transfection cells were cultured in 6-wellplates in suspension with a 50:50 mixture of ADSC complete medium(StemPro™-43) and embryonic stem cells medium (mTeSR1™, StemCellTechnologies). After two days in culture, cells were re-transfected withthe same plasmids and plated in 96 well-plates coated with Matrigel™ (BDBiosciences) in the presence of mTeSR1™ complete medium supplemenetedwith thiazovivin (0.5 μM), an ALK-5 inhibitor (SB341542, Stemgent, 2μM), and an inhibitor of MEK (PD0325901, Stemgent, 0.5 μM). Medium waschanged every day and cells were cultured for 22 days at 37° C., 5% CO₂,5% O₂, followed by AP staining and immunohistochemistry to analyse theexpression of pluripotency markers. Cells formed colonies and were foundto express both pluripotency markers Oct4 and AP after transfectingcells with pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP.

FIG. 17 is a panel showing photographs of ADSCs transiently transfectedwith pCMV6-XL5-Rex1/pCMV6-XL5-Klf4 and pCMV6-XL5-Rex1/pCMV6-XL4-Oct4.After the second transfection, ADSCs were cultured in 96-well platescoated with Matrigel™ for 24 days in the presence of mTeSR1™ mediumsupplemented with SB341542 and PD0325901 at 37° C., 5% CO₂, 5% O₂. Inorder to characterize subpopulations of cells after transfection, livestaining, immunohistochemistry and AP staining were used. 1-5% of totalcells transfected with Rex1/Oct4 or Rex1/Klf4 showed a SSEA-4⁺ andTRA-1-81⁺ phenotype (early pluripotency markers). The observation overtime showed that the phenotype of these colonies moves from an earlySSEA-4⁺ phenotype to a late Oct4⁺/Sox2/Nanog⁺ phenotype starting at Day22, which was closer to the final reprogrammed state and apluripotent-like cell.

FIG. 18 is a panel showing photographs of ADSCs transiently transfectedwith various pluripotent vectors. Following transfection the cells wereplated in StemPro™ MSC SFM medium on Matrigel™ (BD Biosciences) coated24 well plates and incubated at 37° C., 5% CO₂ 5% O₂. On day 1, mediawas changed to a mix of 75% StemPro™ MSC and 25% hES cell medium; thepercentage of StemPro™ MSC SFM medium was decreased every day over fourdays to have 100% hES cell medium by day 4. Then medium was changedevery two days. The hES cell medium consisted in Dulbecco's ModifiedEagle's Medium (DMEM, Invitrogen) supplemented with 20% Knockout™ SerumReplacement (KSR, Invitrogen), 1 mM GlutaMAX™, 100 μM Non-essentialAmino acids, 100 μM β-mercaptoethanol and 10 ng/ml Fgf-2. In order tocharacterize subpopulations of cells after transfection, live staining,immunohistochemistry and AP staining were used. Transfected cellstransfected with Oct4/UTF1/MBD2, Oct4/Dppa4/MBD2, FoxD3/Dppa4/MBD2,Oct4/FoxD3/Dppa4, and Sox2/FoxD3/UTF1 were positive for SSEA-4⁺,TRA1-60, and TRA-1-81⁺ phenotype (early pluripotency markers) at day 14.

FIG. 19 is a panel showing photographs of transiently transfected HFFs.HFFs were transiently transfected using the Nucleofector® II Device(Lonza) following the procedure described in Example II with theexception that 1 μg of each of the following 3 DNA plasmids was used:pCMV-Oct4nuc-IRES2-Sox2nuc, pCMV-Klf4nuc-IRES2-Cmycnuc andpCMV-Nanognuc-IRES2-Lin28. The cells were pre-treated with or withoutVPA and 5-Aza. Following transfection the cells were plated in thefibroblast medium, supplemented with or without VPA (2 mM) and 5-AZA(2.5 μM) on Matrigel™ (BD Biosciences) coated 6-well plates andincubated at 37° C., 5% CO₂. On Day 1 and 2, media was changed to 100%mTeSR1™ medium (StemCell Technologies) supplemented with or without VPAand 5-AZA. On Day 3 and 6, cells were re-transfected as above and platedon Matrigel™ coated plates in mTeSR1™ medium supplemented with orwithout VPA and 5-AZA. Media was changed daily as above. Medium wassupplemented with Y27632 (Stemgent, 10 μM) from Day 7 to Day 14 topromote viability and clonal expansion of potential reprogrammed cells.Cells were analysed at day 20 using the Alkaline Phosphatase DetectionKit (Millipore) and by immunohistochemistry analysis. Some cells stainedpositive for the pluripotency markers AP, SSEA-4 and TRA-1-81 (similarto Mel2 human embryonic stem cell line (positive control)). These cloneswere obtained only in the condition that did not contain inhibitors(i.e.: VPA and 5-AZA). No clones were observed for the condition treatedwith these inhibitors.

FIG. 20 is a panel showing photographs of transfected NSLCs and BG-01.NSLCs and BG-01 NS were transfected as previously described in ExampleII by two episomal vectors, pEF-Oct4nuc-IRES2-MBD2 (NC1) orpCMV-FoxD3-2A-Oct4-2A-Klf4 (F72). Following transfection cells werecollected and plated onto uncoated petri-dishes in the presence ofProliferation medium and mTeSR1™ medium (50:50) into proliferationconditions at 37° C., 5% CO₂. After 48 hours, cells were re-transfectedby the same plasmid and plated in 96-well plates coated with Matrigel™and cultured in the presence of mTeSR1™ medium supplemented by the smallmolecules BIX01294 (Stemgent, 2 μM) and BayK8644 (Stemgent, 2 μM) at 37°C., 5% O₂ for 22 days, after which live staining andimmunohistochemistry were performed to characterize subpopulations ofcells for pluripotency markers. Cells formed colonies positive for bothTRA-1-81 and SSEA-4 indicative of pluripotent-like cells.

FIG. 21 is a panel showing bright field pictures at day 17 offibroblasts transfected with Msi1/Ngn2 and pCMV6-XL5-MBD2 placed indifferent media conditions and showing different morphologies and degreeof differentiation. (a) Cells in neural proliferation medium from day 1to day 12, and then in neural differentiation medium with cytokines fromday 12 to 17. (b) Cells in neural proliferation medium from day 1 to day12, and then in NbActive4 medium with cytokines from day 12 to 17. (c)Cells in neural differentiation medium with cytokines plus Fgf-2 fromday 1 to day 12, and then in the same medium but without Fgf-2 from day12 to 17. (d) Cells in NbActive4 medium with cytokines plus Fgf-2 fromday 1 to day 12, and in then the same medium but without Fgf-2 from day12 to 17. (e) Cells in CDM II medium with cytokines plus Fgf-2 from day1 to day 12, and in then the same medium but without Fgf-2 from day 12to 17.

FIG. 22 is a panel showing pictures of immunochemistry results at day 17of fibroblasts transfected with Msi1/Ngn2 and pCMV6-XL5-MBD2 in FIG. 21.FIGS. 22A and 22B: Cells were in NS-A Proliferation Medium from day 1 today 12, and then in NS-A Differentiation Medium (A) or NBActive4 medium(B) with cytokines from day 12 to 17. There were more cells in B, butDifferentiation from day 12-17 was too short to induce expression ofβIII-tubulin in both cases. FIGS. 22C-E: Cells were in NS-ADifferentiation Medium (C) or NbActive4 medium (D) from day 1-17 (withFGF-2 supplementation from day 1-12), or CDM II medium from day 1-12 andthen NS-A Differentiation Medium from day 12-17 (E). There were a largenumber of cells in C and a much smaller number of cells in D and E.Cells were immunopositive for both GFAP and βIII-tubulin in all casesand placing the cells in differentiation or non-proliferation media fromday 1 onwards appears to have induced a more direct transformation intoneurons and glia, with more intense βIII-tubulin than GFAP positivecells in E.

FIG. 23 is a panel showing two heat maps providing a global overview ofthe gene expression comparison between either NSLC vs. HFF (Set 1), orNSLC vs. hNPC (Set 2). NSLC has a distinct gene expression profile whencompared to either HFF or hNPC. Based on the intensity (the higher theintensity, the higher the relative change in expression), NSLC is muchmore similar to hNPC than to HFF.

FIG. 24 is a panel showing pictures of NSLCs. NSLCs were tested todetermine if they are a population of Skin-Derived Precursors Cells(SKPs). SKPs capable of proliferating in response to EGF and bFGF,express nestin and fibronectin, and can differentiate into both neuronaland mesodermal progeny including into adipocytes. For this purpose astandard protocol for turning SKPs into adipocytes was performed, inwhich adipocyte-derived stem cells (ADSCs) and NSLCs were cultured inStemPro™ proliferation medium and differentiation towards adipocyteswere induced by culturing these cells in differentiation mediumconsisting in DMEM/F12 (50:50), ITS (1:100), HEPES (1:100), GlutaMAX™(1:100), T3 (0.2 nM), Rosiglitasone (0.5 μg/ml), IBMX (1000) andDexamethasone (1 μM). Three days later, IBMX and Dexamethasone werewithdrawn from the medium. At day 10, cells were fixed with a 4%formaldehyde solution for 10 min and stained with Oil Red O (Invitrogen)staining solution. Adipose cells appeared red with lipid droplets(bright white spots in left picture) specifically stained with Oil RedO; however NSLCs stained negative and had no presence of lipid dropletin the cells, but instead adopted neuronal cell morphology. Theseresults conform that NSLCs are not a population of Skin-DerivedPrecursors Cells (SKPs).

FIG. 25 is a panel showing pictures of ADSCs transiently transfectedwith two pluripotent vectors: pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP (NF10) orpCMV-Sall4-2A-Oct4-2A-Klf4-2A-Nanog (S71) using nucleofector asdescribed in Example XIX. Following the transfection cells were culturedin suspension with 50:50 ratio of adipocyte complete medium (StemPro-43)and embryonic stem cells medium (mTesR1, StemCell Technologies). Aftertwo days in culture, cells were re-transfected with the same plasmidslisted above and cells were plated in 96 well-plates coated withMatrigel (BD Biosciences) in the presence of mTesR complete mediumsupplemented with thiazovivin (0.5 .mu.M), an ALK-5 inhibitor (SB341542, Stemgent, 2 .mu.M), and inhibitor of MEK (PD0325901, Stemgent,0.5 .mu.M). Medium was changed every day and cells were cultured for 22days at 37° C., 5% CO₂, 5% O₂. Immunohistochemistry was performed toidentify cells stained with different pluripotency markers. Cells formedcolonies starting day 15 and were found to express pluripotency markers(Sox2, Oct4, Nanog, SSEA, TRA-1-60, TRA-1-81, E-cadherin).

FIG. 26 is a panel of immunofluorescence images illustrating thedifferentiation of PLSCs into ectoderm, mesoderm and endoderm lineages.Representative immunofluorescence images of embryonic bodies derived atday 12, 18, and 28 stained for stage neural markers (13111 tubulin andGFAP), for early mesoderm marker (Brachyury and GATA4); and for earlyendoderm marker (FoxA2).

FIG. 27 is a panel of images comparing the level of expression ofselected genes between NSLC (human neural stem-like cells created fromHFF), NP (human primary neural progenitor cells (Lonza)), FF (the humanforeskin fibroblasts used to create the NSLC (ATCC)), NT2 (human NT2/D1teratocarcinoma cell line (ATCC)), NT2-N (human NT2 neuronsdifferentiated from the NT2/D1 cells), NT2-A (human NT2 astrocytesdifferentiated from the NT2/D1 cells), and NTC no template control forRT-PCR (only primers, no cDNA). (a): Stem Cell Markers: RT-PCR analysisshows that NSLCs express SOX2 but not OCT4a or NANOG. NT2 cells wereused as positive control for SOX2, OCT4 and NANOG. NP cells were used aspositive control for SOX2. (Note: OCT4a is a stem cell marker. OCT4b isa splicing variant of OCT4, which its expression is not limited to stemcells.) (b): Notch Signalling Pathway: NOTCH regulates cell fatedetermination, neuronal differentiation and glial determination. RT-PCRdata show that various components of Notch signalling pathway areexpressed in NSLCs. HES: hairy enhancer of split (a Notch target gene).(c): Early Neuronal Markers. NES: nestin; CD133: also known as Prominin1 (PROW); PAX6: paired box 6; ASCL1: achaete-scute complex homolog 1(also knows as MASH1). (d): Neuron Markers. NFL: neurofilament lightchain; NFM: neurofilament medium chain; NFH: neurofilament heavy chain;SYN: synaptophysin; NSE: neuron-specific enolase; MAP2: microtubuleassociated protein 2a+2b. (e): Neurotrophic factors. BDNF: brain-derivedneurotrophic factor; GDNF: glial cell-derived neurotrophic factor. (f):Astroglial markers. GFAP: glial fibrillary acidic protein. (g): Control.ACTB: beta actin.

FIG. 28 is a panel of immunofluorescence images illustrating functionalcell-cell communication in NSLC cells through gap junctions. A singleNSLC cell was pre-loaded with two dyes [Dil (red) and calcein AM(green)] and plated on an unlabeled layer of NSLC cells. Calcein readilytransferred from the donor cells to a large population of receivingcells after 3 hours in vitro, confirming gap junctional intercellularcommunication among NSLC cells. Calcein AM (MW=994.7 kDa) coverts tocalcein (MW=623 kDa) through estrase hydrolysis after it enters thecell. Calcein passes through gap junctions from donor cell to receivingcells. In contrast, Dil (a cationic membrane tracer used to label thedonor cell) does not pass through gap junctions. (A) red filter; (B)green filter; (C) phase filter; (D) red+green filters; (E) red+phasefilters; and (F) red+green+phase filters.

FIG. 29. Panel (A) shows a light micrograph of NSLCs. Panels (B), (C)and (D) are a panel of immunofluorescence images illustrating that NSLCsreadily express synaptotagmin (a synaptic vesicle protein; panel C) andMap2 (a neural differentiation associated marker; panel B). Panel D is acomposite image of panel B and C.

FIGS. 30 (A) and (B) are a panel of immunofluorescence imagesillustrating that NSLCs readily attach to PGA scaffolds designed forimplantation into the brain. Carboxyfluourodiacetate (CFDA; green stain)confirms optimal cell survival (>93%) 24 hours after cells were platedon the scaffold. NSLC cells showed robust attachment and survival to thesurface for at least one week in vitro. Furthermore, Neurites grow alongthe length of each PGA fiber, following the pattern of the scaffold.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for cell dedifferentiation andcell reprogramming. A significant aspect of the present invention isthat it permits the use of a patient's own cells to develop differenttypes of cells that can be transplanted after steps of in vitrodedifferentiation and in vitro reprogramming. Thus, this technologyeliminates the problems associated with transplantation of non-hostcells, such as, immunological rejection and the risk of transmittingdisease. In addition, since the cells are “newly created”, they have thepotential to be more potent than alternative sources of natural cellsthat have already divided multiple times.

DEFINITIONS

As used herein and in the appended claims, the singular forms “a,” “an”,and “the”, include plural referents unless the context clearly indicatesotherwise. Thus, for example, reference to “a cell” includes one or moreof such cells or a cell line derived from such a cell, reference to “anagent” includes one or more of such agent, and reference to “the method”includes reference to equivalent steps and methods known to those ofordinary skill in the art that could be modified or substituted for themethods described herein.

As used herein, the term “polynucleotide” refers to any DNA or RNAsequence or molecule, comprising encoding nucleotide sequences. The termis intended to encompass all polynucleotides whether occurring naturallyor non-naturally in a particular cell, tissue or organism. This includesDNA and fragments thereof, RNA and fragments thereof, cDNAs andfragments thereof, expressed sequence tags, artificial sequencesincluding randomized artificial sequences.

As used herein, the term “polypeptide” refers to any amino acid sequencehaving a desired functional biological activity (e.g. DNAdemethylation). The term is intended to encompass complete proteins,fragments thereof, fusion proteins and the like, including carbohydrateor lipid chains or compositions.

“Trans-differentiation” refers to a direct switch of an alreadydifferentiated cell to another type of differentiated cell.

“De-differentiation” refers to the loss of phenotypic characteristics ofa differentiated cell by activating or deactivating genes or metabolicpathways.

“Marker” refers to a gene, polypeptide, or biological function that ischaracteristic of a particular cell type or cellular phenotype.

“Genetically-engineered DNA sequence” is meant a DNA sequence whereinthe component sequence elements of DNA sequence are organized within theDNA sequence in a manner not found in nature.

“Signal sequence” refers to a nucleic acid sequence which, whenincorporated into a nucleic acid sequence encoding a polypeptide,directs secretion of the translated polypeptide from cells which expresssaid polypeptide, or allows the polypeptide to readily cross the cellmembrane into a cell. The signal sequence is preferably located at the5′ end of the nucleic acid sequence encoding the polypeptide, such thatthe polypeptide sequence encoded by the signal sequence is located atthe N-terminus of the translated polypeptide. By “signal peptide” ismeant the peptide sequence resulting from translation of a signalsequence.

“Ubiquitous promoter” refers to a promoter that drives expression of apolypeptide or peptides encoded by nucleic acid sequences to whichpromoter is operably linked. Preferred ubiquitous promoters includehuman cytomegalovirus immediate early (CMV); simian virus 40 earlypromoter (SV40); Rous sarcoma virus (RSV); or adenovirus major latepromoter.

“Gene expression profiling” means an assay that measures the activity ofmultiple genes at once, creating a global picture of cellular function.For example, these profiles can distinguish between human neural stemcells and somatic cells that are actively dividing or differentiating.

“Transfection” refers to a method of gene delivery that introduces aforeign nucleotide sequences (e.g. DNA molecules) into a cell preferablyby a non-viral method. In preferred embodiments according to the presentinvention foreign DNA is introduced to a cell by transient transfectionof an expression vector encoding a polypeptide of interest, whereby theforeign DNA is introduced but eliminated over time by the cell andduring mitosis. By “transient transfection” is meant a method where theintroduced expression vectors and the polypeptide encoded by the vector,are not permanently integrated into the genome of the host cell, oranywhere in the cell, and therefore may be eliminated from the host cellor its progeny over time. Proteins, polypeptides, or other compounds canalso be delivered into a cell using transfection methods.

“Neuroprogenitor Cell” refers to an immature cell of the nervous system,which can differentiate into neurons and glia (oligodendrocytes andastrocytes). “Neural Stem Cell” is an ectoderm germ layer derivedmultipotent stem cell having, as a physiological feature, a capacity toform neuroprogenitor cells and under physiological conditions that favordifferentiation to form neurons and glia. “Neural Stem-Like Cell” or“NSLC” refers to any cell-derived multipotent stem cell having, as aphysiological feature, a capacity to form other neural stem-like cellsand neuroprogenitor-like cells and under physiological conditions thatfavor differentiation to form neuron-like cells and glial-like cells.

“Neurosphere” refers to a cellular aggregate of neural stem cells andneuroprogenitor cells that form a floating sphere formed as a result ofproliferation of the neural stem cells and neuroprogenitor cells inappropriate proliferation conditions. NSLCs also form neurospheresconsisting of aggregates of NSLCs and neuroprogenitor-like cells.

“Reprogrammed cell” refers to a cell that has undergone stabletrans-differentiation, de-differentiation, or transformation. Somereprogrammed cells can be subsequently induced to re-differentiate. Thereprogrammed cell stably expresses a cell-specific marker or set ofmarkers, morphology, and/or biological function that was notcharacteristic of the original cell. “Reprogrammed somatic cell” refersto a process that alters or reverses the differentiation status of asomatic cell, which can be either complete or partial conversion of thedifferentiated state to an either less differentiated state or a newdifferentiated state.

“Regeneration” refers to the capability of contributing to the repair orde novo construction of a cell, tissue or organ.

“Differentiation” refers to the developmental process of lineagecommitment of a cell. Differentiation can be assayed by measuring anincrease in one or more cell-differentiation specific markers relativeto the expression of the undifferentiated cell markers.

“Lineage” refers to a pathway of cellular development, in which a moreundifferentiated cell undergoes progressive physiological changes tobecome a more differentiated cell type having a characteristic function(e.g., neurons and glia are of a neuroprogenitor lineage, which is of anectoderm lineage which formed from blastocysts and embryonic stem (ES)cells).

“Tissue” refers to an ensemble of cells (identical or not) and anextracellular matrix (ECM) that together carry out a specific functionor set of functions.

“CDM” is meant a living tissue equivalent or matrix, a living scaffold,or cell-derived matrix.

Cell Transformation

Some aspects of the invention concerns methods and cells to transform orreprogram a given somatic cell into a pluripotent, multipotent and/orunipotent cell. Some aspects of the invention relates to methods forconditioning a somatic cell to reprogramming into a pluripotent,multipotent or unipotent cell.

The terms “transform” or “reprogram” are used interchangeably to referto the phenomenon in which a cell is dedifferentiated ortransdifferentiated to become pluripotent, multipotent and/or unipotent.The dedifferentiated cell could subsequently be redifferentiated into adifferent type of cell. Cells can be reprogrammed or converted tovarying degrees. For example, it is possible that only a small portionof cells are converted or that an individual cell is reprogrammed to bemultipotent but not necessarily pluripotent. Thus, the terms“transforming” or “reprogramming” methods can refer to methods whereinit is possible to reprogram a cell such that the “new” cell showsmorphological and functional characteristics of a new or differentspecific cell lineage (e.g. the transformation of fibroblast cells intoneuronal cells).

As used herein, the term “somatic cell” refers to any differentiatedcell forming the body of an organism, apart from stem cells, progenitorcells, and germline cells (i.e. ovogonies and spermatogonies) and thecells derived therefrom (e.g. oocyte, spermatozoa). For instance,internal organs, skin, bones, blood, and connective tissue are all madeup of somatic cells. Somatic cells according to the invention can bedifferentiated cells isolated from adult or can be fetal somatic cells.Somatic cells are obtained from animals, preferably human subjects, andcultured according to standard cell culture protocols available to thoseof ordinary skill in the art.

As used herein, “Stem cell” refers to those cells which retain theability to renew themselves through mitotic cell division and which candifferentiate into a diverse range of specialized cell types. Itincludes both embryonic stem cells that are found in blastocysts, andadult stem cells that are found in adult tissues. “Totipotent cells”refers to cells that have the ability to develop into cells derived fromall three embryonic germ layers (mesoderm, endoderm and ectoderm) and anentire organism (e.g., human being if placed in a woman's uterus in thecase of humans). Totipotent cells may give rise to an embryo, the extraembryonic membranes and all post-embryonic tissues and organs. The term“pluripotent” as used herein is intended to mean the ability of a cellto give rise to differentiated cells of all three embryonic germ layers.“Multipotent cells” refers to cells that can produce only cells of aclosely related family of cells (e.g. hematopoietic stem cellsdifferentiate into red blood cells, white blood cells, platelets, etc.).“Unipotent cells” refers to cells that have the capacity todevelop/differentiate into only one type of tissue/cell type (e.g. skincells).

The present invention allows the reprogramming of any cell to adifferent type of cell. Although the present application focusesprimarily on the preparation of Stem-Like cells, especially, NeuralStem-Like Cells (NSLCs), the invention is not so restricted because manydifferent types of cells can be generated according to the principlesdescribed herein. Similarly, while the Examples section describesembodiments where fibroblasts, keratinocytes, CD34⁺ cells,adipose-derived stem cells (ADSCs), neural stem cells (including NSLCs),and cells within a Cell-Derived Matrix (CDM) are reprogrammed, theinvention is not limited such cells. The invention may be employed forthe reprogramming of virtually any cell of interest.

Accordingly, a general aspect of the invention relates to a method oftransforming a cell of a first type to a cell of a second differenttype. As used herein, examples of cells of a first type include, but arenot limited to germ cells, embryonic stem cells and derivations thereof,adult stem cells and derivations thereof, progenitor cells andderivations thereof, cells derived from mesoderm, endoderm or ectoderm,and a cell of mesoderm, endoderm or ectoderm lineage such as anadipose-derived stem cell (ADSC), mesenchymal stem cell, hematopoieticstem cell (CD34⁺ cell), skin derived precursor cell, hair follicle cell,fibroblast, keratinocyte, epidermal cell, endothelial cell, epithelialcell, granulosa epithelial cell, melanocyte, adipocyte, chondrocyte,hepatocyte, lymphocyte (B and T lymphocyte), granulocyte, macrophage,monocyte, mononuclear cell, pancreatic islet cell, sertoli cell, neuron,glial cell, cardiac muscle cell, and other muscle cell.

As used herein, examples of cells of a second type include, but are notlimited to germ cells, embryonic stem cells and derivations thereof,adult stem cells and derivations thereof, progenitor cells andderivations thereof, cells derived from mesoderm, endoderm or ectoderm,and a cell of mesoderm, endoderm or ectoderm lineage such as anadipose-derived stem cell, mesenchymal stem cell, hematopoietic stemcell, skin derived precursor cell, hair follicle cell, fibroblast,keratinocyte, epidermal cell, endothelial cell, epithelial cell,granulosa epithelial cell, melanocyte, adipocyte, chondrocyte,hepatocyte, lymphocyte (B and T lymphocyte), granulocyte, macrophage,monocyte, mononuclear cell, pancreatic islet cell, sertoli cell, neuron,glial cell, cardiac muscle cell, and other muscle cell. In addition,each of the above “-like” cell (a cell that has similar but notcompletely identical characteristics of the known natural type of thecell) is also included in the examples of cells of a second type.

According to one particular aspect, the method of transforming a cell ofa first type into a cell of a second different type comprises the stepsof:

-   -   i) providing a cell of a first type;    -   ii) transiently increasing in the cell of a first type        intracellular levels of at least one reprogramming agent,        whereby the transient increase induces direct or indirect        endogenous expression of at least one gene regulator;    -   iii) placing the cell in conditions for supporting the        transformation of the desired cell and maintaining intracellular        levels of the at least one reprogramming agent for a sufficient        period of time to allow stable expression of the at least one        gene regulator in absence of the reprogramming agent; and    -   iv) maintaining the cell in culture conditions supporting the        transformation of the desired cell for a sufficient period of        time to allow a stable expression of a plurality of secondary        genes whose expression is characteristic of phenotypical and        functional properties of the desired cell. At least one of the        stably expressed secondary genes is not characteristic of        phenotypical and functional properties of an embryonic stem        cell. At the end of said period of time the cell of the first        type has been transformed into the desired cell of a different        type. Preferably, the cell of a different type obtained after        the transformation is further characterized by a stable        repression of a plurality of genes expressed in the first cell        type.

According to various embodiments, step iii) may be carried outconsecutively to step ii), simultaneously with step ii), or before stepii).

According to a related aspect, the invention relates to a processwherein a cell of a first type is reprogrammed to a desired cell of adifferent type, the process comprising:

-   -   a transient increase of intracellular levels of at least one        reprogramming agent, wherein the at least one reprogramming        agent induces a direct or indirect endogenous expression of at        least one gene regulator, wherein the endogenous expression of        the at least one gene regulator is necessary for the existence        of the desired cell of a different type;    -   a stable expression of said at least one gene regulator;    -   stable expression of a plurality of secondary genes, wherein the        stable expression of the plurality of secondary genes is the        result of the stable expression of the at least one gene        regulator, and wherein: (i) stable expression of the plurality        of secondary genes is characteristic of phenotypical and/or        functional properties of the desired cell, (ii) stable        expression of at least one of the secondary genes is not        characteristic of phenotypical and functional properties of an        embryonic stem cell, and wherein (i) and (ii) are indicative of        successful reprogramming of the cell of the first type to the        desired cell of the different type.

As used herein, “transiently increasing” refers to an increase that isnot necessarily permanent and therefore, which may decrease or disappearover time. For instance, when referring to transiently increasingintracellular levels of at least one reprogramming agent in a cell, itmeans that the increase in present for a sufficient period of time forcausing particular cellular events to occur (e.g. inducing stableendogenous expression of a gene regulator). Typically a transientincrease is not permanent and is not associated for instance to genomeintegration of an expression vector.

As used herein the term “reprogramming agent” refers to a compound thatis capable of inducing directly or indirectly the expression ofmorphological and/or functional characteristics of the desired cell of adifferent type. Preferred compounds include those capable of drivingdirectly or indirectly transformation of the cell of the first type intothe desired cell of a different type. In preferred embodiment, thereprogramming agent is selected for inducing a direct or indirectendogenous expression of at least one gene regulator as defined herein.There are many compounds that may be helpful in reprogramming a cellaccording to the invention and these compounds can be used alone or incombinations. In various embodiments, the reprogramming agent is apolynucleotide or polypeptide selected according to TABLE A:

TABLE A Reprogramming agent Examples of RefSeq/ UniProt ™/ Desired CellGenBank ™ (NCBI) Swiss-Prot UniGene ™ Type Name Access. No. Access. No.Accession No. Markers Pluripotent- AGR2 NM_006408.3 O95994 Hs.530009OCT4 like cells AGR3 NM_176813.3 Q8TD06 Hs.100686 Nanog BRIX1NM_018321.3 Q8TDN6 Hs.718510 SSEA-4 CRABP2 NM_001878.2 P29373 Hs.405662TRA1-60 DNMT3B, isoform 1 NM_006892.3 Q9UBC3 Hs.713611 TRA1-80 DNMT3B,isoform 2 NM_175848.1 Q9UBC3 Hs.713611 AP DNMT3B, isoform 3 NM_175849.1Q9UBC3 Hs.713611 DNMT3B, isoform 6 NM_175850.1 Q9UBC3 Hs.713611 DPPA2NM_138815.3 Q7Z7J5 Hs.351113 DPPA3 (STELLA) NM_199286.2 Q6W0C5 Hs.131358DPPA4 NM_018189.3 Q7L190 Hs.317659 DPPA5 (ESG1) NM_001025290.1 A6NC42Hs.125331 FOXD3 NM_012183.2 Q9UJU5 Hs.546573 FOXH1 NM_003923.2 O75593Hs.708365 GABRB3, isoform 1 NM_000814.5 P28472 Hs.302352 GABRB3, isoform2 NM_021912.4 P28472 Hs.302352 GABRB3, isoform 3 NM_001191320.1 P28472Hs.302352 GABRB3, isoform 4 NM_001191321.1 P28472 Hs.302352 GBX2NM_001485.2 P52951 Hs.184945 GDF3 NM_020634.1 Q9NR23 Hs.86232 GJA1(CX43) NM_000165.3 P17302 Hs.74471 GRB7 NM_005310.2 Q14451 Hs.86859NM_001030002.1 Q14451 Hs.86859 HESRG NR_027122.1 Q1W209 Hs.720658 IFITM1NM_003641.3 P13164 Hs.458414 IFITM2 NM_006435.2 Q01629 Hs.709321 KLF2NM_016270.2 Q9Y5W3 Hs.726356 KLF4 NM_004235.4 O43474 Hs.376206 LEFTY1NM_020997.2 O75610 Hs.656214 LEFTY2 (EBAF), NM_003240.3 O00292 Hs.520187isoform 1 LEFTY2 (EBAF), NM_001172425.1 B4E332 Hs.520187 isoform 2(TrEMBL) LIN28A NM_024674.4 Q9H9Z2 Hs.86154 MYBL2 NM_002466.2 P10244Hs.179718 NANOG NM_024865.2 Q9H9S0 Hs.635882 NODAL NM_018055.4 Q96S42Hs.370414 NOG NM_005450.4 Q13253 Hs.248201 NR0B1 (DAX1) NM_000475.4P51843 Hs.268490 NR5A2, isoform 1 NM_205860.1 O00482 Hs.33446 NR5A2,isoform 2 NM_003822.3 O00482 Hs.33446 NR6A1, isoform 1 NM_033334.2Q15406 Hs.586460 NR6A1, isoform 2 NM_001489.3 Q15406 Hs.586460 PHC1NM_004426.2 P78364 Hs.305985 PITX2, isoform a NM_153427.1 Q99697Hs.643588 PITX2, isoform b NM_153426.1 Q99697 Hs.643588 PITX2, isoform cNM_000325.5 Q99697 Hs.643588 PODXL, isoform 1 NM_001018111.2 O00592Hs.726449 PODXL, isoform 2 NM_005397.3 O00592 Hs.726449 POU5F1 (OCT4),NM_002701.4 O01860 Hs.249184 isoform 1* POU5F1 (OCT4), NM_203289.4 N/AHs.249184 isoform 2 NM_001173531.1 PTEN NM_000314.4 P60484 Hs.500466REST NM_005612.4 Q13127 Hs.307836 NM_001193508.1 Q13127 Hs.307836 REX1NM_020695.3 Q8N1G1 Hs.192477 SALL4 NM_020436.3 Q9UJQ4 Hs.517113 SEMA3ANM_006080.2 Q14563 Hs.252451 SFRP2 NM_003013.2 Q96HF1 Hs.481022 SOX2NM_003106.2 P48431 Hs.518438 TDGF1, isoform 1 NM_003212.3 P13385Hs.385870 TDGF1, isoform 2 NM_001174136.1 P13385 Hs.385870 TERT, isoform1 NM_198253.2 O14746 Hs.492203 TERT, isoform 2 NM_001193376.1 O14746Hs.492203 TPT1 NM_003295.2 P13693 Hs.374596 UTF1 NM_003577.2 Q5T230Hs.458406 ZFP42 NM_174900.3 Q96MM3 Hs.335787 Ectoderm- ASCL1 (MASH1)NM_004316.3 P50553 Hs.703025 FoxJ3 like cells CDX1 NM_001804.2 P47902Hs.1545 Otx2 DLX3 NM_005220.2 O60479 Hs.134194 E-cadherin DLX5NM_005221.5 P56178 Hs.99348 TP73L FOXD3 NM_012183.2 Q9UJU5 Hs.546573MSI1 NM_002442.2 O43347 Hs.158311 NANOG NM_024865.2 Q9H9S0 Hs.635882POU5F1 (OCT4), NM_002701.4 Q01860 Hs.249184 isoform 1* POU5F1 (OCT4),NM_203289.4 N/A Hs.249184 isoform 2 NM_001173531.1 SOX1 NM_005986.2O00570 Hs.202526 SOX2 NM_003106.2 P48431 Hs.518438 SP8, isoform 1NM_182700.4 Q8IXZ3 Hs.195922 SP8, isoform 2 NM_198956.2 N/A Hs.195922ZIC1 NM_003412.3 Q15915 Hs.647962 Mesendoderm- EOMES NM_005442.2 O95936Hs.591663 Mixl1 like cells FOXA2, isoform 1* NM_021784.4 Q9Y261Hs.155651 Mesp1 FOXA2, isoform 2 NM_153675.2 Q9Y261 Hs.155651 Bry FOXD3NM_012183.2 Q9UJU5 Hs.546573 Flk1 GATA4 NM_002052.3 P43694 Hs.243987Pax2 GATA6 NM_005257.3 Q92908 Hs.514746 Six1 MIXL1 NM_031944.1 Q9H2W2Hs.282079 POU5F1 (OCT4), NM_002701.4 Q01860 Hs.249184 isoform 1* POU5F1(OCT4), NM_203289.4 N/A Hs.249184 isoform 2 NM_001173531.1 SOX17NM_022454.3 Q9H6I2 Hs.98367 T (Brachyury) NM_003181.2 O15178 Hs.389457Desired second cell type Neural stem- CALB1 NM_004929.2 P05937 Hs.65425Sox2 like cells DLL1 NM_005618.3 O00548 Hs.379912 Nestin DLX1, isoform 1NM_178120.4 P56177 Hs.407015 GFAP DLX1, isoform 2 NM_001038493.1 P56177Hs.407015 Msi1 DLX2 NM_004405.3 Q07687 Hs.419 Sox1 FOXD3 NM_012183.2Q9UJU5 Hs.546573 CD133 GJD2 (CX36) NM_020660.1 Q9UKL4 Hs.283816 HES1NM_005524.2 Q14469 Hs.250666 HES3 NM_001024598.3 Q5TGS1 Hs.532677 HES5NM_001010926.3 Q5TA89 Hs.57971 HOXB1 NM_002144.3 P14653 Hs.99992 MNX1(HB9), NM_005515.3 P50219 Hs.37035 isoform 1 MNX1 (HB9), NM_001165255.1N/A Hs.37035 isoform 2 MSI1 NM_002442.2 O43347 Hs.158311 NANOGNM_024865.2 Q9H9S0 Hs.635882 NEUROD1 NM_002500.2 Q13562 Hs.709709NEUROG1 NM_006161.2 Q92886 Hs.248149 NEUROG2 NM_024019.2 Q9H2A3Hs.567563 NKX6.1 NM_006168.2 P78426 Hs.546270 PAX6, isoform a*NM_000280.3 P26367 Hs.270303 PAX6, isoform a NM_001127612.1 P26367Hs.270303 PAX6, isoform b NM_001604.4 P26367 Hs.270303 SFRP2 NM_003013.2Q96HF1 Hs.481022 SIX3 NM_005413.3 O95343 Hs.567336 SOX1 NM_005986.2O00570 Hs.202526 SOX2 NM_003106.2 P48431 Hs.518438 Cardiac BAF60C(SMARCD3), NM_001003802.1 Q6STE5 Hs.647067 MLc2α progenitor- isoform 1like cells BAF60C (SMARCD3), NM_003078.3 Q6STE5 Hs.647067 Nkx2.5 isoform1 BAF60C (SMARCD3), NM_001003801.1 Q6STE5 Hs.647067 Isl+ isoform 2*FOXD3 NM_012183.2 Q9UJU5 Hs.546573 Bry GATA4 NM_002052.3 P43694Hs.243987 GATA6 NM_005257.3 Q92908 Hs.514746 HAND1 NM_004821.2 O96004Hs.152531 HAND2 NM_021973.2 P61296 Hs.388245 ISL1 NM_002202.2 P61371Hs.505 KDR NM_002253.2 P35968 Hs.479756 MESP1 NM_018670.3 Q9BRJ9Hs.447531 MYOCD, isoform 1 NM_001146312.1 Q6N065 Hs.567641 (TrEMBL)MYOCD, isoform 2 NM_153604.2 Q8IZQ8 Hs.567641 MYOCD, isoform 3NM_001146313.1 Q8IZQ8 Hs.567641 NKX2.5, isoform 1* NM_004387.3 P52952Hs.54473 NKX2.5, isoform 2* NM_001166175.1 P52952 Hs.54473 NKX2.5,isoform 3* NM_001166176.1 P52952 Hs.54473 T (Brachyury) NM_003181.2O15178 Hs.389457 TBX5, isoform 1* NM_000192.3 Q99593 Hs.381715 TBX5,isoform 1 NM_181486.1 Q99593 Hs.381715 TBX5, isoform 2 NM_080718.1Q99593 Hs.381715 TBX5, isoform 3 NM_080717.2 Q99593 Hs.381715 SOX17NM_022454.3 Q9H6I2 Hs.98367 Pancreatic FOXA2, isoform 1* NM_021784.4Q9Y261 Hs.155651 PDX1 progenitor- FOXA2, isoform 2 NM_153675.2 Q9Y261Hs.155651 Sox17 like cells FOXD3 NM_012183.2 Q9UJU5 Hs.546573 FoxA2 MAFANM_201589.2 Q8NHW3 Hs.670866 Ngn3 MIXL1 NM_031944.1 Q9H2W2 Hs.282079Isl1 NEUROG3 NM_020999.3 Q9Y4Z2 Hs.532682 NKX6.1 NM_006168.2 P78426Hs.546270 PAX4 NM_006193.2 O43316 Hs.129706 PDX1 NM_000209.3 P52945Hs.32938 SOX17 NM_022454.3 Q9H6I2 Hs.98367 Myogenic FOXC1 NM_001453.2Q12948 Hs.348883 SMα actin progenitor- FOXC2 NM_005251.2 Q99958Hs.436448 Calponin like cells MEF2C, isoform 1 NM_002397.4 Q06413Hs.649965 MyoD NM_001193350.1 Q06413 MEF2C, isoform 2 NM_001131005.2Q06413 Hs.649965 MEF2C MEF2C, isoform 3 NM_001193347.1 Q06413 Hs.649965Pax3 MEF2C, isoform 4 NM_001193348.1 Q06413 Hs.649965 Pax7 MEF2C,isoform 5 NM_001193349.1 Q06413 Hs.649965 Pax3, isoform Pax3 NM_181457.3P23760 Hs.42146 Pax3, isoform Pax3a NM_000438.5 P23760 Hs.42146 Pax3,isoform Pax3b NM_013942.4 P23760 Hs.42146 Pax3, isoform Pax3dNM_181458.3 Q494Z3, Q494Z4 Hs.42146 (TrEMBL) Pax3, isoform Pax3eNM_181459.3 Q494Z3, Q494Z4 Hs.42146 (TrEMBL) Pax3, isoform Pax3gNM_181461.3 Q494Z3, Q494Z4 Hs.42146 (TrEMBL) Pax3, isoform Pax3hNM_181460.3 Q494Z3, Q494Z4 Hs.42146 (TrEMBL) Pax3, isoform Pax3iNM_001127366.2 Q494Z4 Hs.42146 (TrEMBL) PAX7, isoform 1 NM_002584.2P23759 Hs.113253 PAX7, isoform 2 NM_013945.2 P23759 Hs.113253 PAX7,isoform 3 NM_001135254.1 P23759 Hs.113253

In some embodiments, the reprogramming agent is a polypeptide whichshares at least 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of thefunctionality or sequence identity of any one of the reprogrammingagents in the table hereinbefore.

Identifying the “sufficient period of time” to allow stable expressionof the at least one gene regulator in absence of the reprogramming agentand the “sufficient period of time” in which the cell is to bemaintained in culture conditions supporting the transformation of thedesired cell is within the skill of those in the art. The sufficient orproper time period will vary according to various factors, including butnot limited to, the particular type and epigenetic status of cells (e.g.the cell of the first type and the desired cell), the amount of startingmaterial (e.g. the number of cells to be transformed), the amount andtype of reprogramming agent(s), the gene regulator(s), the cultureconditions, presence of compounds that speed up reprogramming (ex,compounds that increase cell cycle turnover, modify the epigeneticstatus, and/or enhance cell viability), etc. In various embodiments thesufficient period of time to allow a stable expression of the at leastone gene regulator in absence of the reprogramming agent is about 1 day,about 2-4 days, about 4-7 days, about 1-2 weeks, about 2-3 weeks orabout 3-4 weeks. In various embodiments the sufficient period of time inwhich the cells are to be maintained in culture conditions supportingthe transformation of the desired cell and allow a stable expression ofa plurality of secondary genes is about 1 day, about 2-4 days, about 4-7days, or about 1-2 weeks, about 2-3 weeks, about 3-4 weeks, about 4-6weeks or about 6-8 weeks. In preferred embodiments, at the end of thetransformation period, the number of transformed desired cells issubstantially equivalent or even higher than an amount of cells a firsttype provided at the beginning.

The present invention encompasses various types of compounds that aresuitable for increasing in a cell of a first type the intracellularlevels of at least one reprogramming agent. Preferably, the compoundshould also be able to directly or indirectly remodel the chromatinand/or DNA of the cell, thus resulting directly or indirectly in theexpression of morphological and functional characteristics of thedesired cell of a different type. Preferred compounds are reprogrammingagents as defined herein or any other compound having a similar activityand having the ability to activate or enhance the expression of theendogenous version of genes listed in the table of reprogramming agentshereinbefore and which are capable of driving directly or indirectlytransformation of the cell of the first type into the desired cell of adifferent type.

As will be explained hereinafter, the increase in intracellular levelsof the at least one reprogramming agent can be achieved by differentmeans. In preferred embodiments the reprogramming agent is a polypeptideand increasing intracellular levels of such polypeptide includetransfection (or co-transfection) of an expression vector having apolynucleotide (ex. DNA or RNA) encoding the polypeptide(s), or by anintracellular delivery of polypeptide(s). According to the invention,transient expression is generally preferable. Additional suitablecompounds may include compounds capable of increasing the expression ofthe endogenous version of genes listed in the table of reprogrammingagents and gene regulators including, but not limited to, reprogrammingfactors listed in Table B.

TABLE B Desired cell of different type Reprogramming FactorPluripotent-like cells Nodal, ActivinA, Fgf-2, Wnt3a, L-Ascorbic Acid,BIO, CHIR99021, PD0325901, Thiazovivin, SB431542, Cyclic Pifithrin-α,Tranylcypromine hydrochloride, Kenpaullone, 5-Azacytidine, ValproicAcid, BIX01294, R(+)BayK8644, RG108, Theanine, Sodium butyrate Ectodermlike cells: a retinoid compound, L-Ascorbic acid, SHH, Wnt 3a, a 1-Neural stem-like cells neurotrophic factor, bFGF, EGF, Transforminggrowth factor alpha, neuropeptide Y, Estrogen, Noggin, Forskolin, 5-Azacytidine, Valproic Acid, BIX01294, R(+)BayK8644, RG108, Sodiumbutyrate, Lithium Mesoendoderm like cells: BMP4, Epidermal growthfactor-Cripto/FRL-1/Cryptic (EGF-CFC) and the TGFβs, Activin, Nodal,SHH, Vg1/GDF1 (growth and differentiation factor-1) 1- Cardiacprogenitor-like 1- BMP4, bFGF, Activin A, VEGF, DKK1 (dickkopf cellshomologue 1), Insulin-like growth factor 1 (IGF-1) and hepatocyte growthfactor (HGF), 5-Azacytidine, Valproic Acid, BIX01294, R(+)BayK8644,RG108, Cardiogenol C hydrochloride, Sodium butyrate 2- Pancreaticprogenitor- 2- Activin A, GLP-1, bFGF, Reg1, nicotinamide, like cellsBetacellulin, SHH, (−)-Indolactam V, a retinoid compound, Cyclopamine,IDE-1 and 2, 5-Azacytidine, Valproic Acid, BIX01294, R(+)BayK8644,RG108, Sodium butyrate 3- Myogenic progenitor- 3- retinoic acid, HGF,FGF, IGF, transforming growth like cells factor-beta, Wnt3a,5-Azacytidine, Valproic Acid, BIX01294, R(+)BayK8644, RG108, Sodiumbutyrate

According to the principles of the invention, increasing intracellularlevels of at least one reprogramming agent should induce a direct orindirect endogenous expression of at least one gene regulator. As usedherein, “gene regulator” refers to a polynucleotide or polypeptide whoseexpression is associated with a series of intracellular events leadingto the transformation of a given cell of a first type into apluripotent, multipotent and/or unipotent cell. Typically expression ofa gene regulator directly or indirectly activates genes necessary forthe phenotypical and functional characteristic of pluripotent,multipotent and/or unipotent cells, while repressing genes of the cellof a first type. The gene regulator may be the same or be different thanthe reprogramming agent. Examples of gene regulators according to theinvention include, but are not limited to, the polynucleotides andpolypeptides listed herein before in TABLE A.

In some embodiments, the gene regulator is a polypeptide which shares atleast 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of the functionality orsequence identity of any one of the gene regulators provided in theTable A hereinbefore.

As used herein, “conditions supporting growth” or “conditions supportingthe transformation” when referring to a desired cell refers to varioussuitable culture conditions (temperature, pH, O₂ tension, cell media,factors, compounds, growth substrate (ex. laminin, collagen,fibronectin, Matrigel™, low-bind surface, nanostructured or chargedsurface, etc.), 3D environment, etc.) favorising growth of the desiredcell type and/or favorising transformation towards such desired celltype. Those skilled in the art known that growth or transformation ofparticular cell types is stimulated under specific conditions, whileinhibited by others, and it is within their skill to select suitableconditions (e.g. culture conditions) favorising growth or transformationof desired cell types.

The terms “phenotypical and functional properties”, when referring to adesired cell or to an embryonic stem cell, means the biological,biochemical, physiological and visual characteristics of a cell,including expression of certain genes and cell surface markers, whichcan be measured or assessed for confirming its identity or function(s).

An example of a suitable reprogramming agent according to preferredembodiments of the invention is MUSASHI1. In some embodiments thispolypeptide is preferred for driving a first cell, such as a fibroblast,into a Neural Stem-Like Cell (NSLC). In other embodiments, the at leastone reprogramming agent which said intracellular levels is increasedis(are) either Musashi1 (Msi1) alone; Musashi1 (Msi1) and Neurogenin 2(Ngn2); Musashi1 (Msi1) and methyl-CpG binding domain protein 2 (MBD2);or Neurogenin 2 (Ngn2) and methyl-CpG binding domain protein 2 (MBD2).Adequate intracellular levels of these polypeptides are preferred sincethey tend to be expressed throughout an entire cell lineage, from asearly as embryonic stem cells (or even earlier) to pre-somatic cells (oreven later).

MBD2 is a member of a family of methyl-CpG-binding proteins that hasbeen reported to be both a transcriptional repressor and a DNAdemethylase (dMTase). As used herein, the term “MBD2” generally refersto the human methyl-CpG binding domain protein 2. The GeneBank™ (NCBI)accession number of human MBD2 is NM_003927.3/AF072242, the UniProt™accession number is NP-003918/Q9UBB5 and the UniGene™ accession numberis Hs.25674.

As used herein, the term “Msi1” generally refers to the human musashihomolog 1. The GeneBank™ (NCBI) accession number of human Msi1 isNM_002442.2/AB012851, the UniProt™ accession number is NP-002433/043347and the UniGene™ accession number is Hs.158311.

As used herein, the term “Ngn2” generally refers to the human neurogenin2. The GeneBank™ (NCBI) accession number of human Ngn2 isNM_024019.2/BC036847, the UniProt™ accession number is NP-076924/Q9H2A3and the UniGene™ accession number is Hs.567563.

According to additional aspects, the method of transforming a cell of afirst type to a desired cell of a different type comprises the steps ofeither:

-   -   1) contacting the cell of a first type with one or more        compounds capable of increasing intracellular levels of at least        one reprogramming agent within the cell and directly or        indirectly remodeling the chromatin and/or DNA of the cell; or    -   2) contacting the chromatin and/or DNA of a cell of a first type        with an agent capable of remodeling the chromatin and/or DNA of        the cell; and increasing intracellular levels of at least one        reprogramming agent.

According to various embodiments, step 2) may be carried outconsecutively to step 1), simultaneously with step 1), or before step1).

According to a particular aspect, the invention relates to a method forobtaining a Neural Stem-Like Cell (NSLC), comprising:

-   -   providing a cell of a first type which is not a NSLC;    -   increasing intracellular levels of at least one neural stem cell        specific polypeptide, wherein the polypeptide is capable of        driving directly or indirectly transformation of the cell of the        first type into a NSLC; and    -   contacting chromatin and/or DNA of a cell of a first type with a        histone acetylator, an inhibitor of histone deacetylation, a DNA        demethylator, and/or a chemical inhibitor of DNA methylation.

With respect to the second step, the term “remodelling the chromatinand/or DNA” refers to dynamic structural changes to the chromatin. Thesechanges can range from local changes necessary for transcriptionalregulation, to global changes necessary for opening up the chromatinstructure or chromosome segregation to allow transcription of the newset of genes characteristic of the desired cell of a different type, toclosing up of the chromatin structure or chromosome segregation toprevent transcription of certain genes that are not characteristic ofthe desired cell of a different type. In some embodiments, opening up ofthe chromatin structure refers more specifically to acetylation ofhistones, and demethylation of DNA, while closing up of the chromatinstructure refers more specifically to deacetylation of histones, andmethylation of DNA.

As used herein, “compound” refers to a compound capable of effecting adesired biological function. The term includes, but is not limited to,DNA, RNA, protein, polypeptides, and other compounds including growthfactors, cytokines, hormones or small molecules. As used herein,compounds capable of remodeling chromatin and/or DNA include, but arenot limited to, histone acetylators, inhibitors of histonedeacetylation, DNA demethylators, inhibitors of DNA methylation andcombination thereof.

“Inhibitor of DNA methylation” refers to an agent that can inhibit DNAmethylation. DNA methylation inhibitors have demonstrated the ability torestore suppressed gene expression. Suitable agents for inhibiting DNAmethylation include, but are not limited to 5-azacytidine,5-aza-2-deoxycytidine, 1-β-D-arabinofuranosil-5-azacytosine, anddihydro-5-azacytidine, and zebularine (ZEB), BIX (histone lysinemethyltransferase inhibitor), and RG108.

“Inhibitor of histone deacetylation” refers to an agent that preventsthe removal of the acetyl groups from the lysine residues of histonesthat would otherwise lead to the formation of a condensed andtranscriptionally silenced chromatin. Histone deacetylase inhibitorsfall into several groups, including: (1) hydroxamic acids such astrichostatin (A), (2) cyclic tetrapeptides, (3) benzamides, (4)electrophilic ketones, and (5) aliphatic acid group of compounds such asphenylbutyrate and valporic acid. Suitable agents to inhibit histonedeacetylation include, but are not limited to, valporic acid (VPA),phenylbutyrate Trichostatin A (TSA), Na-butyrate, and benzamides. VPApromotes neuronal fate and inhibits glial fate simultaneously throughthe induction of neurogenic transcription factors including NeuroD.

“Histone Acetylator” refers to an agent that inserts acetyl groups tothe lysine residues of histones that opens up the chromatin and turns itinto a transcriptionally active state. Suitable Histone Acetylatoragents include, but are not limited to, Polyamine, CREB (cAMP elementbinding protein), and BniP3.

“DNA demethylator” refers to an agent that removes the methyl groupsfrom DNA and possesses the ability to inhibit hypermethylation andrestore suppressed gene expression. A demethylase is expected toactivate genes by removing the repressive methyl residues. Suitable DNAdemethylators include, but are not limited to, MBD2 and Gadd45b.

In some embodiments, the reprogramming agent has one or more of thefollowing functions: it decrease the expression of one or more markersof cells of the first type (ex. see Table C), and/or increase theexpression of one or more markers of the desired cell of the differenttype (ex. see Table A). Cells that exhibit a selectable marker for thedesired cell of a different type are then selected and assessed forcharacteristics of the desired cell of a different type.

According to the invention, transformation into the desired cell resultsin stable expression of a plurality of secondary genes whose expressionis characteristic of phenotypical and/or functional properties of thedesired cell. Genes whose expression is characteristic of phenotypicaland/or functional properties of the desired cell include, but is notlimited to, those listed in Table A.

In some embodiments, expression of secondary genes whose expression ischaracteristic of phenotypical and functional properties of the desiredcell results in the expression of markers defined according to thefollowing table:

Desired cell type Markers Neural stem-like cells Nestin, Sox2, GFAP,Msi1 Neural-like cells βIII-tubulin, Map2b, Synapsin, ACHE Ectoderm-likecells Sox2, Sox1, Zic1, Nestin, Notch 1, FoxJ3, Otx2, Cripto1, VimentinMesendoderm-like cells Sox17, FoxA2, CXCR4, GATA4, Mixl1, EomesoderminPluripotent-like cells Oct4, SSEA4, TRA-1-60, TRA-1-81, AP

In some embodiments, transformation of a cell of a first type into thedesired cell results in a stable repression of a plurality of genestypically expressed in the cell of the first type. Examples of suchsuppressed genes include, but are not limited to, those defined in TableC:

TABLE C Examples of suppressed genes Cell-type specific genes typicallyrepressed during Reprogramming RefSeq/ UniProt ™/ GenBank ™ (NCBI)Swiss-Prot UniGene ™ Cell Type Name Accession No. Accession No.Accession No. Markers Keratinocytes TP63, NM_003722.4 Q9H3D4 Hs.137569Keratin 14 isoform 1 TP63, NM_001114978.1 Q9H3D4 Hs.137569 Basonuclinisoform 2 TP63, NM_001114979.1 Q9H3D4 Hs.137569 P63 isoform 3 TP63,NM_001114980.1 Q9H3D4 Hs.137569 isoform 4 TP63, NM_001114981.1 Q9H3D4Hs.137569 isoform 5 TP63, NM_001114982.1 Q9H3D4 Hs.137569 isoform 6 BNC1NM_001717.3 Q01954 Hs.459153 BCN2 NM_017637.5 Q6ZN30 Hs.656581 KRT14NM_000526.4 P02533 Hs.654380 Involucrin NM_005547.2 P07476 Hs.516439Fibroblasts THY1 NM_006288.3 P04216 Hs.724411 Col5A2 FBN2 NM_001999.3P35556 Hs.519294 Fibronectin COL5A2 NM_000393.3 P05997 Hs.445827 DNMT1,NM_001130823.1 P26358 Hs.202672 isoform a DNMT1, NM_001379.2 P26358Hs.202672 isoform b CD34+ Isl1 NM_002202.2 P61371 Hs.505 VEGFR HOXA9NM_152739.3 P31269 Hs.659350 Cytokeratin HOXB4 NM_024015.4 P17483Hs.664706 Klk-1 NM_002257.2 P06870 Hs.123107 CD34 Bry NM_003181.2 O15178Hs.389457 Adipose- ALCAM NM_001627.2 Q13740 Hs.591293 ALBO derived stemVCAM-1 NM_001078.2 P19320 Hs.109225 Adiponectin cells (ADSC) VCAM-1,NM_080682.1 P19320 Hs.109225 isoform b PROM1, NM_006017.2 O43490Hs.614734 Leptin isoform 1 PROM1, NM_001145847.1 O43490 Hs.614734isoform 2 NM_001145848.1 PROM1, NM_001145852.1 O43490 Hs.614734 isoform4 PROM1, NM_001145851.1 O43490 Hs.614734 isoform 5 PROM1, NM_001145850.1O43490 Hs.614734 isoform 6 PROM1, NM_001145849.1 O43490 Hs.614734isoform 7 FUT4 NM_002033.3 P22083 Hs.390420

In preferred embodiments, stable repression of any one or more of thegenes listed in Table C being expressed in the first cell type is alsocharacterized by a disappearance of the corresponding markers (see TableC).

Those skilled in the art will understand that there exist manyalternative steps for facilitating cell reprogramming. Those includedestabilizing the cell's cytoskeletal structure (for example, byexposing the cell to cytochalasin B), loosening the chromatin structureof the cell (for example, by using agents such as 5 azacytidine (5-Aza)and Valproic acid (VPA) or DNA demethylator agents such as MBD2),transfecting the cell with one or more expression vector(s) containingat least one cDNA encoding a neurogenic transcription factor (forexample, Msi1 or Ngn2), using an appropriate medium for the desired cellof a different type and an appropriate differentiation medium to inducedifferentiation commitment of the desired cell of a different type,inhibiting repressive pathways that negatively affects induction intocommitment the desired cell of a different type, growing the cells on anappropriate substrate for the desired cell of a different type (forexample, laminin for NSLCs or a low-bind surface for culturing floatingneurospheres), and growing the cells in an environment that the desiredcell of a different type (or “-like” cell) would be normally exposed toin vivo such as the proper temperature, pH and low oxygen environment(for example about 2-5% O₂). In various embodiments, the inventionencompasses these and other related methods and techniques forfacilitating cell reprogramming.

Accordingly, the method of transforming a cell of a first type into acell of a second different type may comprise additional facultativesteps. In one embodiment, the method of transforming a cell furthercomprises the step of pretreating the cell of a first type with acytoskeleton disruptor. As used herein “cytoskeleton” refers to thefilamentous network of F-actin, Myosin light and heavy chain,microtubules, and intermediate filaments (IFs) composed of one of threechemically distinct subunits, actin, tubulin, or one of several classesof IF protein. Accordingly, the term “cytoskeleton disruptor” refers toany molecules that can inhibit the cell cytoskeleton to destabilize thecell and consequently remove the feedback mechanisms between the cell'sshape and cellular and nuclear function. Suitable cytoskeleton disruptoraccording to the invention include, but are not limited to, thecytochalasin family of actin cytoskeleton inhibitors, such asCytochalasin B or D, and myosin inhibitors such as 2,3-butanedionemonoxime. Such pretreatment may boost reprogramming. In a preferredembodiment, the cell is cultured in the presence of at least onecytoskeleton inhibitor one day before, during, or after introducing aneurogenic transcription factor(s).

Placing the cell in conditions in conditions for supporting thetransformation of the desired cell, and/or maintaining the cell inculture conditions supporting the transformation of the desired cell maycomprises culturing the cell in a media comprising one or more factorsappropriate for inducing the expression of the morphological andfunctional characteristics of the desired desired cell of a differenttype. In some embodiments the one or more factors are reprogrammingfactors helpful in reprogramming a cell and these reprogramming factorscan be used alone or in combinations.

In other embodiments, the step of culturing the cell in a mediacomprising one or more factors appropriate for inducing the expressionof the morphological and functional characteristics of the desired cellof a different type is carried out subsequently or simultaneously tosteps iii) or iv), or subsequently or simultaneously to steps 1) or 2),as defined hereinbefore.

Those skilled in the art know many different types of media and manyreprogramming factors that may be helpful in reprogramming a cell andthese reprogramming factors can be used alone or in combinations. Invarious embodiments, the reprogramming factor is selected according toTABLE B.

In some embodiments, reprogramming factors have one or more of thefollowing functions: decrease the expression of one or more markers ofthe first type of cell and/or increase the expression of one or moremarkers of the desired cell. Cells that exhibit a selectable marker forthe desired cell are then selected and assessed for unipotency,multipotency, pluripotency, or similar characteristics (as appropriate).

In particular embodiments, the cells are cultured in serum-free mediumbefore, during or after any one of steps i) to iv) as definedhereinbefore, or during or after steps 1) or 2), as definedhereinbefore.

In some embodiments, Mesendoderm-like cells can be created from cellssuch as ADSCs by up-regulating the expression of FoxD3, MixI1, Ngn3 andMBD2. Additionally up-regulating the expression of Oct4, Sox17,Brachyury, and/or FoxA2 can be used to create Mesendoderm-like cells. Inaddition, media compositions detailed in Table 37 can be used to createmesendoderm-like cells from adipocyte-derived stem cells (ADSCs) andsimilar cells, along with the use of proper cell substrates formesendoderm cells known in the art (e.g., gelatin-coated plates).

In some embodiments, cells such as ADSCs can be reprogrammed intopancreatic progenitor-like cells and β islet-like cells by up-regulatingthe expression of Sox17, Pdx1 Ngn3, and Oct4 (e.g.,Oct4+Sox17+Pdx1+Ngn3; Sox17+Pdx1+Ngn3; Oct4+Pdx1+Ngn3). Additionallyup-regulating the expression of FoxA2 and MBD2 can be used to createthese cells. In addition, media compositions detailed in Table 39 can beused to create pancreatic progenitor-like cells and β islet-like cellsfrom adipocyte-derived stem cells (ADSCs) and similar cells, along withthe use of proper cell substrates for growing pancreatic progenitorcells and β islet-like cells known in the art (e.g., fibronectin-coatedcollagen gels).

In some embodiments, cells such as ADSCs can be reprogrammed intocardiac progenitor-like cells and mesoderm-like cells by up-regulatingthe expression of a combination of Mesp1, FoxD3, Tbx5, Brachyury (T),Nkx2.5, Sox17 and/or Gata4 (e.g., Foxd3+Sox17+Mesp1+Tbx5;Foxd3+Sox17+Mesp1+Nkx2.5; Foxd3+T+Mesp1+Gata4), which increase theexpression of mesoderm and cardiac progenitors markers. Additionallyup-regulating the expression of Gata6 and Baf60c can be used to createthese cells. In addition, media compositions detailed in Table 44 can beused to create cardiac progenitor-like cells from adipocyte-derived stemcells (ADSCs) and similar cells, along with the use of proper cellsubstrates for growing cardiac progenitor cells known in the art (e.g.,matrigel).

In some embodiments, cells such as ADSCs can be reprogrammed intopluripotent-like stem cells up-regulating the expression of Rex1, Oct4and Klf4, or Sall4, Oct4, Klf4 and Nanog. Reprogramming by transienttransfection of a combination of the above genes can be achievedefficiently. These cells can be differentiated into ectoderm-like cells,endoderm-like cells, or mesendoderm-like cells by methods known in theart for differentiating pluripotent stem cells into these lineages. Inaddition, these pluripotent-like stem cells have protective and/ortherapeutic/regenerative effect on other cells (e.g., hepatocytes).

The NSLCs that is a subject of this invention have benefits over nativehuman neural stem/progenitor cells as well as embryonic stem cells. TheNSLCs do not readily form tumors or teratomas (as tested in NOD-SCIDmice), can be created from easily obtainable somatic cells (e.g.,fibroblasts, OD34+ blood cells, keratinocytes) and differentiatedtowards any specific neuronal lineage, express neurotrophic growthfactors (e.g., BDNF and GDNF), express some neuronal differentiationgenes while maintaining a stem cell like state allowing a higherproportion of neuronal differentiation when placed in differentiationconditions (compared to native human neural stem/progenitor cells), formfunctional gap junctions and readily form synapses, and are capable ofattaching and surviving on 3D scaffolds and environments.

The stem-like cells of the present invention have numerous advantagesover the prior art such as efficient reprogramming without geneintegration or constant artificial forced gene expression, greaterpotency and safety over native cells (esp. stem/progenitor cells), andhaving the capability to be the cell of interest for a particularapplication (e.g., a neural stem-like cell for CNS applications; acardiac progenitor-like cell for cardiac applications, a pancreaticprogenitor-like cells or (3 islet-like cells for diabetes, anectoderm-like cell, a mesoderm-like cells, an endoderm-like cell, etc.).In addition derived cells of the invention can be a relativelyhomogeneous population of autologous cells of a particular phenotype ofinterest.

The methods of the present invention allow the ability to create thenative types of stem cells for particular applications (e.g., neuralstem-like cells for CNS applications; cardiac progenitor-like cells forcardiac applications, pancreatic progenitor-like cells or .beta.-likecells for diabetes, etc.) as well as creating autologous (from thepatient's own cells) versions of these stem cells that allow them tograft more appropriately when delivered to the patient as a treatment orfor augmenting the health or functionality of a particular tissue ororgan, or for diagnostic purposes. The cells can also be used formodeling (e.g., disease modeling, or testing the effects of particularcompounds or other molecules (e.g., for personalized medicinepurposes)). The cells can also easily be enhanced by specific genes ofinterest or a defective gene repaired/replaced before delivery to thepatient for treatment of a genetic disorder or for enhanced therapeuticvalue or augmenting the health or functionality of a particular cell,tissue, organ, system, or organism.

Obtaining Neural Stem-Like Cells (NSLCs)

According to preferred embodiments for creating Neural Stem-Like Cells(NSLCs), the methods of the invention are carried out such that cellsare treated with selected agents, compounds and factors to promote thereprogramming and/or dedifferentiation towards Stem-Like Cells (SLCs).Such reprogrammed somatic cells can then be further treated with agentsand/or cultured under conditions suitable for promoting reprogrammingtowards Neural Stem-Like Cells (NSLCs), and expansion of the NSLCs forthe long-term. NSLCs according to the invention have the potential todifferentiate to neuronal-like and/or glial-like cells, as well asneuronal and/or glial cells, for potential treatment of neurologicaldiseases and injuries such as Parkinson's disease and spinal cordinjury. The methods described herein are also useful for producinghistocompatible cells for cell therapy.

Accordingly, some aspects of the present invention relates to generatingneurons from an individual patient, thus making autologoustransplantations possible as a treatment modality for many neurologicalconditions including neurotrauma, stroke, neurodegenerative diseasessuch as Multiple Sclerosis, Parkinson's disease, Huntington disease,Alzheimer's diseases. Thus, the invention provides for neurologicaltherapies to treat the disease or trauma of interest.

Therefore, another aspect of the invention concerns a method ofobtaining a Neural Stem-Like Cell (NSLC), comprising either:

-   -   1) contacting the cell of a first type with one or more neural        stem cell regulating polypeptide capable of increasing        intracellular levels of neural stem cell specific polypeptides        within said cell and directly or indirectly remodeling the        chromatin and/or DNA of the cell and driving directly or        indirectly transformation of the cell of the first type into a        NSLC; or    -   2) contacting the chromatin and/or DNA of a cell of a first type        with a histone acetylator, an inhibitor of histone        deacetylation, a DNA demethylator, and/or an inhibitor of DNA        methylation; and increasing intracellular levels of at least one        neural stem cell specific polypeptide driving directly or        indirectly transformation of the cell of the first type into a        NSLC.

In preferred embodiments, the step 1) comprises increasing intracellularlevels of a MUSASHI1 polypeptide. As it will be explained hereinafterthis can be achieved by different means including, but not limited to,transient expression of the MUSASHI1 polypeptide, preferably bytransfecting an expression vector encoding the polypeptide.

In preferred embodiments, the step 2) comprises increasing intracellularlevels of a MBD2 polypeptide or treating the cells with VPA and 5-AZA.As it will be explained hereinafter this can be achieved by differentmeans including, but not limited to, transient expression of the MBD2polypeptide, preferably by transfecting an expression vector encodingthe polypeptide(s), and/or pre-treating and/or treating the cells withVPA and 5-AZA.

In one particular embodiment, reprogramming a cell of a first type toanother type of cell that exhibits at least two selectable markers forneural stem cells requires transfecting the cell of a first type withone vector containing a cDNA encoding for a neurogenic transcriptionfactor and one DNA demethylator. To enhance the de-differentiation thecells are exposed or pre-exposed to an agent(s) that inhibits DNAmethylation, inhibits histone deacetylation, and/or disrupts the cellcytoskeleton. For example, the dedifferentiation can be enhanced bypre-treating the cells with an agent that disrupts the cell cytoskeletonfollowed by transfecting the cells with one or more vector(s) containingtwo neurogenic transcription factors in the presence of a DNAdemethylator and/or inhibitor of DNA methylation and histonedeacetylation. The histone deacetylator, inhibitor of histonedeacetylation, DNA demethylator, and/or an inhibitor of DNA methylationare as defined previously.

As defined previously, the method may further comprise a preliminarystep of pre-treating the cell of a first type with a cytoskeletondisruptor, as defined previously, and/or culturing the cell in a mediacomprising one or more reprogramming factors appropriate for appearanceand maintenance of the morphological and functional characteristics ofNSLCs as defined previously (e.g. a retinoid compound, a neurotrophicfactor, bFGF, EGF, SHH, Wnt 3a, neuropeptide Y, Estrogen). In someembodiment the method further comprises inhibiting cellular BMPsignaling pathways (e.g. by NOGGIN, fetuin, or follistatin).

In preferred embodiments, generation of a NSLC from a first cellcomprises the use of one or more reprogramming agents. Suitable agentsinclude, but are not limited to, Musashi-1 (Msi1) and Neurogenin 2(Ngn2). Other potential agents are listed in Table A and B.

The present invention is also directed to the use of DNA expressionvectors encoding a protein or transcript which upregulates theexpression of neurogenesis. The genetically-engineered DNA sequence,encoding a defined reprogramming agent such as Msi1 and Ngn2, can beintroduced into cells by using a mono-, bi-, or poly-cistronic vectors.The expression of an endogenous multipotency gene indicates that thecDNA encodes a protein whose expression in the cell result directly orindirectly in the de-differentiation of the cell. The newlyde-differentiated mammalian cells are capable of re-differentiating toneuronal lineages to regenerate said mammalian cells, tissues, andorgans.

The present invention is further directed to a method for generatingNSLCs by introducing a genetically-engineered DNA sequence into humansomatic cells via transient transfection. Since the DNA introduced inthe transfection process is not inserted into the nuclear genome, theforeign DNA decreases over time and when the cells undergo mitosis.Nonviral vectors remain in a non-replicative form, have lowimmunogenicity, and are easy and safe to prepare and to use.Furthermore, plasmids may accommodate large fragments of DNA.

In one particular embodiment, the method starts with obtaining cellsfrom the individual, and reprogramming the cells in vitro to generateNSLCs. The significant aspect of the present invention is the stablereprogramming of a somatic cell or non-neuronal cell into a NSLC thatcan give rise to different types of, neuronal or glial cells (includingneuronal-like or glial-like cells). These can then be implanted backinto the same patient from which the cells were obtained, thus making anautologous treatment modality for many neurological conditions includingneurotrauma, stroke, and neurodegenerative disease possible. These canalso be implanted into a different individual from which the cells wereobtained. Accordingly, the cells and methods of the present inventionmay be helpful to treat, prevent, or to stabilize a neurological diseasesuch as Alzheimer's disease, Parkinson's disease, multiple sclerosis, orspinal cord injury. This technology provides an ample source of neuralstem cells, neuro-progenitor cells, neurons and glia for clinicaltreatment, which can be performed by implantation of NSLCs in vivo orinducing the differentiation in vitro and implantation ofneuro-progenitor cells or specific neurons or glia in vivo.

In another embodiment, the method comprises isolating somatic ornon-neuronal cells and exposing the cells to one or more agents thatalter cell morphology and chromatin structure, and transfecting thecells with one or more genes containing at least one cDNA encoding for aneurogenic transcription factor. The gene transfection step may bereplaced with alternative agents that induce the expression of theneurogenic transcription factor(s) in the cell. Inducing epigeneticmodifications to DNA and histones (especially DNA demethylation and anopen chromatic structure) facilitate true reprogramming of the cells. Inanother embodiment, the cells are incubated in a low oxygen environment,for example 5% O₂, thereby helping in reprogramming the cells.

This methodology allows the reprogramming of a cell into a NSLC. Thefurther course of development and the expansion of the reprogrammed celldepend on the in situ environment cues to which it is exposed. Theembodiments of the invention further include growing the reprogrammedcell in an appropriate proliferation medium to expand the generatedNSLC, for example Neural Progenitor proliferation Medium (StemCellTechnologies) with the presence of epidermal growth factor (EGF) andbasic fibroblast growth factor (bFGF), to promote the neural stem cellto proliferate.

The NSLCs obtained according to the invention can be differentiated intoneuronal, astrocyte, and/or oligodendrocyte lineages in appropriatedifferentiation medium, for example NS-A differentiation medium(StemCell, Technologies) or NbActive medium (BrainBits™) including aretinoid compound, such as all-trans-retinoic acid or vitamin A, andBDNF, to induce the differentiation of NSLCs towards neuronal and/orglial cells. Neuronal cells include cells that display one or moreneural-specific morphological, physiological, functional and/orimmunological features associated with a neuronal cell type. Usefulcriteria features includes: morphological features (e.g., long processesor neurites), physiological and/or immunological features such asexpression of a set of neuronal-specific markers or antigens, synthesisof neurotransmitter(s) such as dopamine or gamma aminobutyric acid(GABA), and functional features such as ion channels or actionpotentials characteristic of neurons.

In accordance with the method, reprogrammed cells can be selected basedon differential adherence properties as compared to untransfected cells;for example, reprogrammed cells can form floating neurospheres or growwell on laminin while untransfected fibroblasts attach and grow well onregular cell culture treated plates. Reprogrammed cells include cellsthat exhibit one or more neural stem specific markers and morphology andthe loss of some or all of the specific markers related to the originalcells. Furthermore, some of the functionality of the neural-like cells(NLCs) can be assessed at different time points by, for example,patch-clamping, immunostaining for synaptophysin and MAP2b, and byimmunochemical means such as by enzyme-linked immunosorbent assay(ELISA).

In certain embodiments, the present invention provides NSLCs that areable to initiate and direct central nervous system regeneration at asite of tissue damage and can be customized for individual patientsusing their own cells as the donor or starting cell. The presentinvention can be used to generate cells from an individual patient, thusmaking autologous transplantations possible as a treatment modality formany neurological conditions. Thus, this technology eliminates theproblems associated with transplantations of non-host cells, such as,immunological rejection and the risk of transmitted disease. The greatadvantage of the present invention is that it provides an essentiallylimitless supply for autologous grafts suitable for transplantation.Therefore, it will obviate some significant problems associated withcurrent source of materials and methods of transplantation.

Delivery of Polynucleotides

In certain embodiments, the invention concerns the use ofpolynucleotides, e.g. a polynucleotide encoding a MBD2 polypeptide, aMUSASHI1 polypeptide and/or a Ngn2 polypeptide. Means for introducingpolynucleotides into a cell are well known in the art. Transfectionmethods of a cell such as nucleofection and/or lipofection, or othertypes of transfection methods may be used. For instance a polynucleotideencoding a desired polypeptide can be cloned into intermediate vectorsfor transfection in eukaryotic cells for replication and/or expression.Intermediate vectors for storage or manipulation of the nucleic acid orproduction of protein can be prokaryotic vectors, (e.g., plasmids),shuttle vectors, insect vectors, or viral vectors for example. A desiredpolypeptide can also be encoded by a fusion nucleic acid.

To obtain expression of a cloned nucleic acid, it is typically subclonedinto an expression vector that contains a promoter to directtranscription. Suitable bacterial and eukaryotic promoters are wellknown in the art and described, e.g., in Sambrook and Russell (MolecularCloning: a laboratory manual, Cold Spring Harbor Laboratory Press). Thepromoter used to direct expression of a nucleic acid of choice dependson the particular application. For example, a strong constitutivepromoter is typically used for expression and purification. In contrast,when a dedifferentiation protein or compound is to be used in vivo,either a constitutive or an inducible promoter or compound is used,depending on the particular use of the protein. In addition, a weakpromoter can be used, such as HSV TK or a promoter having similaractivity. The promoter typically can also include elements that areresponsive to transactivation, e.g., hypoxia response elements, Ga14response elements, lac repressor response element, and small moleculecontrol systems such as tet-regulated systems and the RU-486 system.

In addition to a promoter, an expression vector typically contains atranscription unit or expression cassette that contains additionalelements required for the expression of the nucleic acid in host cells,either prokaryotic or eukaryotic. A typical expression cassette thuscontains a promoter operably linked, e.g., to the nucleic acid sequence,and signals required, e.g., for efficient polyadenylation of thetranscript, transcriptional termination, ribosome binding, and/ortranslation termination. Additional elements of the cassette mayinclude, e.g., enhancers, and heterologous spliced intronic signals.

Expression vectors containing regulatory elements from eukaryoticviruses are often used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 late promoter, metallothionein promoter, murine mammary tumor viruspromoter, Rous sarcoma virus promoter, polyhedrin promoter, or otherpromoters shown effective for expression in eukaryotic cells.

Standard transfection methods can be used to produce bacterial,mammalian, yeast, insect, or other cell lines that express largequantities of dedifferentiation proteins, which can be purified, ifdesired, using standard techniques. Transformation of eukaryotic andprokaryotic cells is performed according to standard techniques.

Any procedure for introducing foreign nucleotide sequences into hostcells can be used. These include, but are not limited to, the use ofcalcium phosphate transfection, DEAE-dextran-mediated transfection,polybrene, protoplast fusion, electroporation, lipid-mediated delivery(e.g., liposomes), microinjection, particle bombardment, introduction ofnaked DNA, plasmid vectors, viral vectors (both episomal andintegrative) and any of the other well known methods for introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell (see, e.g., Sambrook et al., supra). It isonly necessary that the particular genetic engineering procedure used becapable of successfully introducing at least one gene into the host cellcapable of expressing the protein of choice.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids into mammalian cells or target tissues. Suchmethods can be used to administer nucleic acids encoding reprogrammingpolypeptides to cells in vitro. Preferably, nucleic acids areadministered for in vivo or ex vivo gene therapy uses. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome. Viral vectordelivery systems include DNA and RNA viruses, which have either episomalor integrated genomes after delivery to the cell.

Methods of non-viral delivery of nucleic acids include lipofection,microinjection, ballistics, virosomes, liposomes, immunoliposomes,polycation or lipid-nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection reagents are soldcommercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutrallipids suitable for efficient receptor-recognition lipofection ofpolynucleotides are known. Nucleic acid can be delivered to cells (exvivo administration) or to target tissues (in vivo administration). Thepreparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to those of skillin the art.

The use of RNA or DNA virus-based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, wherein the modifiedcells are administered to patients (ex vivo). Conventional viral basedsystems for the delivery include retroviral, lentiviral, poxviral,adenoviral, adeno-associated viral, vesicular stomatitis viral andherpesviral vectors, althoughntegration in the host genome is possiblewith certain viral vectors, including the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials. In applications for which transient expression ispreferred, adenoviral-based systems are useful. Adenoviral based vectorsare capable of very high transduction efficiency in many cell types andare capable of infecting, and hence delivering nucleic acid to, bothdividing and non-dividing cells. With such vectors, high titers andlevels of expression have been obtained. Adenovirus vectors can beproduced in large quantities in a relatively simple system.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application. Alternatively, vectors can bedelivered to cells ex vivo, such as cells explanted from an individualpatient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) oruniversal donor hematopoietic stem cells, followed by reimplantation ofthe cells into a patient, usually after selection for cells which havebeen reprogrammed.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a nucleicacid (gene or cDNA), and re-infused back into the subject organism(e.g., patient). Various cell types suitable for ex vivo transfectionare well known to those of skill in the art.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic nucleic acids can be also administered directly to theorganism for transfection of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells. Suitable methods of administering such nucleic acids areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Delivery of Polypeptides

In most, if not all the methods described herein, an alternativepossibility consists of bypassing the use of a polynucleotide andcontacting a cell of a first type cell directly with a compound (e.g. apolypeptide) for which an increased intracellular level is desired. Inother embodiments, for example in certain in vitro situations, the cellsare cultured in a medium containing one or more functional polypeptides.

An important factor in the administration of polypeptides is ensuringthat the polypeptide has the ability to traverse the plasma membrane ofa cell, or the membrane of an intra-cellular compartment such as thenucleus. Cellular membranes are composed of lipid-protein bilayers thatare freely permeable to small, nonionic lipophilic compounds and areinherently impermeable to polar compounds, macromolecules, andtherapeutic or diagnostic agents. However, proteins, lipids and othercompounds, which have the ability to translocate polypeptides across acell membrane, have been described. For example, “membrane translocationpolypeptides” have amphiphilic or hydrophobic amino acid subsequencesthat have the ability to act as membrane-translocating carriers.Polypeptides for which an increased intracellular level is desiredaccording to the invention can be linked to suitable peptide sequencesfor facilitating their uptake into cells. Other suitable chemicalmoieties that provide enhanced cellular uptake can also be linked,either covalently or non-covalently, to the polypeptides. Other suitablecarriers having the ability to transport polypeptides across cellmembranes may also be used.

A desired polypeptide can also be introduced into an animal cell,preferably a mammalian cell, via liposomes and liposome derivatives suchas immunoliposomes. The term “liposome” refers to vesicles comprised ofone or more concentrically ordered lipid bilayers, which encapsulate anaqueous phase. The aqueous phase typically contains the compound to bedelivered to the cell.

In certain embodiments, it may be desirable to target a liposome usingtargeting moieties that are specific to a particular cell type, tissue,and the like. Targeting of liposomes using a variety of targetingmoieties (e.g., ligands, receptors, and monoclonal antibodies) has beenpreviously described.

Cells and Cell Lines

The invention encompasses the cells, cell lines, stem cells and purifiedcell preparations derived from any of the methods described herein. Insome embodiments, the cells, cells lines, stem cells and purified cellspreparations of the invention are of mammalian origins, including butnot limited to human, primates, rodent, dog, cat, horse, cow, or sheep.In preferred embodiments, they originate from a human.

Accordingly, another aspect of the invention relates to modified cells,cell lines, pluripotent, multipotent or unipotent cells and purifiedcell preparations, wherein any of these cells comprise an exogenouspolynucleotide encoding Musashi1 (Msi1); Msi1 and Ngn2; Msi1 and MBD2;and Ngn2 and MBD2; Msi1, Ngn2 and MBD2; Msi1, Ngn2, Nestin and MBD2; andother potential combinations from Table A preferably including Msi1 andNgn2 and MBD2. In preferred embodiments the cell according to theinvention is a stem-like cell, more preferably a Neural Stem-Like Cell(NSLC), the cell possessing one or more of the followingcharacteristics:

-   -   expression of one or more neural stem cell marker selected from        the group consisting of Sox2, Nestin, GFAP, Msi1, and Ngn2;    -   decreased expression of one or more genes specific to the cell        that the NSLC was obtained from (e.g. see Table C);    -   forms neurospheres in the neurosphere colony formation assay;    -   capable of being cultured in suspension or as an adherent        culture;    -   capable of proliferating without the presence of an exogenous        reprogramming agent for over 1 month, preferably over 2 months,        over 3 months, over 5 months and even for more than a year;    -   capable of dividing every 36 hours at low passage;    -   positive for telomerase activity;    -   capable of differentiation into a neuronal-like cell,        astrocyte-like cell, oligodendrocyte-like cell and combinations        thereof;    -   decreased expression of telomerase and one or more neural stem        cell markers after differentiation;    -   having one or more morphological neurite-like processes (axons        and/or dendrites) greater than one cell diameter in length after        differentiation into a neuronal-like cell;    -   expression of at least one neural-specific antigen selected from        the group consisting of neural-specific tubulin, microtubule        associated protein 2, NCAM, and marker for a neurotransmitter        after differentiation into a neuronal-like cell;    -   expression of one or more functional neural markers (e.g.        synapsin) after differentiation into a neuronal-like cell;    -   capable of releasing one or more neurotrophic factors (e.g.        BDNF) after differentiation into a neuronal-like cell;    -   negative in a tumor colony forming assay;    -   negative for tumor growth in SCID mice;    -   negative for teratoma growth in SCID mice;    -   capable of significantly improving one or more functional        measures after placement of an adequate number of NSLCs into the        void in a brain ablation model;    -   capable of significantly improving or maintaining one or more        functional measures after injecting an adequate number of NSLCs        into an EAE model; and    -   capable of improving one or more functional measures more        significantly than hNPCs in CNS injury or neurodegenerative        models.

Examples of all of the above items can be found in the Examples sectionof this application.

In preferred embodiments, a NSLC according to the inventions possessesall of the following characteristics:

-   -   ability to self-renew for significantly longer than a somatic        cell;    -   is not a cancerous cell;    -   is stable and not artificially maintained by forced gene        expression or by similar means and may be maintained in standard        neural stem cell media;    -   can differentiate to a progenitor, precursor, somatic cell or to        another more differentiated cell type of the same lineage;    -   has the characteristics of a stem cell and not just certain        markers or gene expression or morphological appearance; and    -   does not exhibit uncontrolled growth, teratoma formation, and        tumor formation in vivo.

In one particular embodiment, the reprogrammed cells (NSLCs) accordingto the invention are capable of proliferating for several months withoutlosing their neural stem cell markers and their ability to differentiatetowards neuron-like, astrocyte-like, and oligodendrocyte-like cells. Thegeneration of the neural lineages is characterized based on morphology,phenotypic changes and functionality.

In some embodiments, the cells of the invention may have one or more ofthe following characteristics and properties: self-renewal, multilineagedifferentiation in vitro and in vivo, clonogenicity, a normal karyotype,extensive proliferation in vitro under well defined culture conditions,and the ability to be frozen and thawed, as well as any of the commonlyknown and/or desired properties or characteristics typical of stemcells. The cells of the invention may further express molecular markersof multipotent or pluripotent cells (i.e. gene and surface markers asdefined previously).

Another aspect of the invention relates to the production of tissuespecific autologous (self) stem and/or progenitor cells. These stemand/or progenitor cells may be used in cell therapy applications totreat diseases of cellular degeneration. Diseases of cellulardegeneration include, for example, neurodegenerative diseases such asstroke, Alzheimer's disease, Parkinson's disease, multiple sclerosis,Amyotrophic lateral sclerosis, macular degeneration, osteolytic diseasessuch as osteoporosis, osteoarthritis, bone fractures, bone breaks,diabetes, liver injury, degenerative diseases, myocardial infarct, burnsand cancer. It is envisioned that cells according to the invention maybe implanted or transplanted into a host. An advantage of the inventionis that large numbers of autologous stem cells can be produced forimplantation without the risk of immune system mediated rejection. Thosecells can lead to production of tissue suitable for transplant into theindividual. Since the tissue is derived from the transplant recipient,it should not stimulate an immune response, as would tissue from anunrelated donor. Such transplants can constitute tissues (e.g. vein,artery, skin, muscle), solid organ transplants (e.g., heart, liver,kidney), neuronal cell transplants, or bone marrow transplants such asare used in the treatment of various malignancies such as, for example,leukemias and lymphomas. Neural stem cell, neuroprogenitor, or neuronalcell (as well as NSLCs and derivations thereof) transplants can also beused in the treatment of, for example, neurological disorders, stroke,spinal cord injury, Parkinson's disease, and the like, as well aspotentially some non-neurological disorders such as a cardiac infarct.

Another aspect of the invention relates to a method to produce ex vivoengineered tissues for subsequent implantation or transplantation into ahost, wherein the cellular components of those engineered tissuescomprise cells according to the invention, or cells derived therefrom.For example, expanded cultures of the cells of the invention may bedifferentiated by in vitro treatment with growth factors and/ormorphogens. Populations of differentiated cells are then implanted intothe recipient host near the site of injury or damage, or cultured invitro to generate engineered tissues, as described.

The methods and cells of the invention described herein can be used toimmortalize cells, for example to generate a cell line. Using themethods disclosed herein, a somatic cell can be transformed into onepossessing a dedifferentiated phenotype, thereby facilitating thegeneration of cell lines from a variety of tissues. Therefore, theinvention encompasses such immortalized cells.

In addition, the methods of deriving the cells according to theinvention, may be helpful in scientific and therapeutic applicationsincluding, but not limited to, (a) scientific discovery and researchinvolving cellular development and genetic research (e.g. uses in lieuof human stem cells as a model cell line to study the differentiation,dedifferentiation, or reprogramming of human cells), (b) drugdevelopment and discovery (e.g., screening for efficacy and toxicity ofcertain drug candidates and chemicals, screening for prospective drugsor agents which mediate the differentiation, dedifferentiation, orreprogramming of cells), (c) gene therapy (e.g., as a delivery devicefor gene therapy), and (d) treatment of injuries, trauma, diseases anddisorders including, but not limited to, Parkinson's, Alzheimer's,Huntington's, Tay-Sachs, Gauchers, spinal cord injury, stoke, burns andother skin damage, heart disease, diabetes, Lupus, osteoarthritis, liverdiseases, hormone disorders, kidney disease, leukemia, lymphoma,multiple sclerosis, rheumatoid arthritis, Duchenne's Musclar Dystrophy,Ontogenesis Imperfecto, birth defects, infertility, pregnancy loss, andother cancers, degenerative and other diseases and disorders.

Additional aspects concern therapeutic methods, methods of treatment andmethods of regenerating a tissue or organ in a mammal (e.g. a humansubject). One particular method concerns a method of regenerating amammalian tissue or organ which comprises contacting the tissue or organto be regenerated with a SLC, NSLC, or other desired cell or artificialtissue construct as defined herein. The SLC, NSLC, desired cell orartificial tissue construct may be placed in proximity to the tissue ororgan to be regenerated by administering to the subject using anysuitable route (e.g. injecting the cell intrathecally, directly into thetissue or organ, or into the blood stream).

Another method for repairing or regenerating a tissue or organ in asubject in need thereof comprises administering to the subject acompound inducing a direct or indirect endogenous expression of at leastone gene regulator in cells of the tissue or organ and/or a compoundinducing a direct or indirect endogenous expression of at least one generegulator in cells capable of transformation or dedifferentiation invivo in the subject. Accordingly, the expression of the at least onegene regulator reprograms the cells into desired cells of a differenttype (e.g. neural stem-like cells), and these cells of a different typeare effective in repairing or regenerating said tissue or organ.

Another method comprises obtaining cells or tissue from a patient (e.g.hematopoietic stem cells, fibroblasts, or keratinocytes), reprogramminga plurality of such cells or the tissue, and reintroducing thereprogrammed cells or tissue into the patient. A related aspect concernspharmaceutical compositions comprising a plurality of a desired cell,SLC and/or Neural Stem-Like Cell (NSLC) or reprogrammed tissue asdefined herein.

The therapeutic methods of the invention may be applicable to theregeneration or repair of various tissues and organs including, but notlimited to, the brain, the spine cord, the heart, the eye, the retina,the cochlea, the skin, muscles, intestines, pancreas (including betacells), kidney, liver, lungs, bone, bone marrow, cartilage, cartilagediscs, hair follicles, teeth, blood vessels, glands (including endocrineand exocrine glands), ovaries, reproductive organs, mammary and breasttissue.

A related aspect concerns pharmaceutical compositions comprising aplurality of desired cell, SLC and/or Neural Stem-Like Cell (NSLC) asdefined herein.

Tissues

Another aspect of the invention relates to a tissue containingreprogrammed cells as defined herein that can be implanted into asubject in need thereof.

In some embodiments the present invention provides for the reprogrammingof cells within a tissue, for example an in vitro produced 3D tissueconstruct comprising cells and extracellular matrix produced by thesecells. In addition, transfected cells can be seeded on top of these 3Dtissue constructs that can be made completely autologously, thuspreventing host rejection, making it completely immunocompatible and ascarrier for reprogrammed cells to be transplanted in vivo.Advantageously, these newly created cells can be used in theirundifferentiated and/or differentiated state within these tissues for invitro diagnostic purposes or transplanted into a patient in need of sucha construct in cell therapy/tissue replacement approaches.

The invention further encompasses 3D Neuronal-Like multilayer tissue.Cells within CDM reprogrammed to Neural Stem-Like Cells according to theinvention readily differentiate into neuronal-like cells, astrocyte-likecells, and oligodendrocyte-like cells within the CDM. It is thuspossible to use CDM and reprogramming methods of the invention toreprogram the cells within the CDM to form 3D Neuronal-Like multilayertissue (up to >30 cell layers). Such 3D tissue comprises neurons (orspecifically, neuron-like cells), astrocytes (or specifically,astrocyte-like cells), and oligodendrocytes (or specifically,oligodendrocyte-like cells) and it can be made completely autologously,can be manually handled and implanted with relative ease, or can used asan in vitro CNS tissue model.

One particular aspect concerns an artificial tissue construct whichcomprises a 3D assembly of in vitro cultured cells and extracellularmatrix produced by these cells. The cells may be desired cells, SLCand/or a plurality of Neural Stem-Like Cell (NSLC) obtained using anyone of the methods described herein.

Screening Methods

Another aspect of the invention relates to methods for identifying newcompounds (e.g. small molecules, drugs, etc) capable of transforming acell of a first type to a desired cell of a different type. These newcompounds may be useful for research purposes or as medicaments for usein repairing or regenerating tissues in a subject.

The Examples section provides principles, methods and techniques usefulfor screening and identifying such desirable active compounds. Forinstance, those skilled in the art will understand that it isconceivable to screen for compounds that will induce transformation of acell of a first type to a NSLC by replacing the “induction” or“biological activity” provided by the transient increase of Musashi1,NGN2 or MBD2 in the cell by a candidate compound to be tested (e.g. alibrary of small molecules or compounds) and measuring activity orefficacy of the candidate compound in generating the NSLC. Individual ormixture of active compounds would be selected if they have the sameactivity and/or if they can provide the same or similar effects as thesepolypeptides (e.g. cell transformation and/or appearance of anydesirable markers or desirable characteristics as defined hereinbefore).For example, a compound or mixture of compounds capable of transforminga fibroblast into a NSLC could be identified by:

-   -   (i) Setting up, culturing and transforming the fibroblasts into        NSLC as in Example 1;    -   (ii) Screening a library of compounds by replacing Msi1, Ngn2        and/or MBD2 with each candidate compound in a different well;    -   (iii) Identify a compound ‘hit’ when the candidate compound is        able to transform the fibroblasts into NSLCs approximately as        well as the replaced Msi1, Ngn2 and/or MBD2;    -   (iv) If compound from part (iii) did not replace all of Msi1,        Ngn2 and MBD2, and is not able to transform the fibroblasts into        NSLCs by itself, then by including the compound from (iii) in        each well, screening a library of compounds by replacing the        Msi1, Ngn2 and/or MBD2 that was not removed in part (ii) with        each candidate compound in a different well;    -   (v) Identify a compound ‘hit’ when the candidate compound is        able to transform, along with the compound from part (iii), the        fibroblasts into NSLCs approximately as well as the replaced        Msi1, Ngn2 and/or MBD2;    -   (vi) If compound from part (V) did not replace all of Msi1, Ngn2        and/or MBD2, and is not able to transform the fibroblasts into        NSLCs together with the compound from part (iii), then by        including the compound from (iii) and (v) in each well,        screening a library of compounds by replacing the Msi1, Ngn2 or        MBD2 that was not removed in part (ii) and (iv) with each        candidate compound in a different well;    -   (vii) Identify a compound ‘hit’ when the candidate compound is        able to transform, along with the compound from part (iii) and        (v), the fibroblasts into NSLCs approximately as well as the        replaced Msi1, Ngn2 or MBD2;    -   (viii) A combination of the compounds from part (iii), (v)        and (vii) will be able to transform the fibroblasts into NSLC;        modifications to these compounds can be made and further        screened to identify more effective or safe versions of these        compounds.

The same principles are applicable for other desired types of stem-likecells including pluripotent-like cells, mesendoderm-like cells,pancreatic progenitor-like cells, etc. Tables A and B, and the Examplessection provides, for each of these types of cells, a list of potentialgenes and/or compounds to be considered in such screening methods.

Accordingly, the present invention encompasses these and any equivalentscreening methods where candidate compounds are tested for theirefficacy in transforming a cell of a first type to a desired cell of adifferent type when compared to the efficacy of the reprogramming factorand/or gene regulator as defined herein.

Delivery of Neurotrophic Factors

Local delivery of neurotrophic factors has been suggested as a method totreat several neurological conditions. Strategies using neurotrophicmolecules focus on preventing the progressive loss of neurons,maintaining neuronal connections and function (neuroprotection), andinducing additional regenerative responses in neurons such as increasedneurotransmitter turnover and/or axonal sprouting (neuroregeneration).Up to date, several therapeutic strategies to deliverneurotrophic-factors in animal models have been explored, but so fartesting of the effects of growth factors on the brain and nervous systemhave been limited to direct peripheral injection of large doses of thesefactors, which carries a significant risk of side effects. Accordingly,a related aspect of the invention relates to overcoming these problemsby using NSLC cells and cell lines according to the invention which canstably express and secrete growth factors of potential interest aftertransplantation.

To summarize, the present invention provides a plentiful source ofNeural Stem-Like Cells, Neuron-Like Cells, Astrocyte-Like Cells orOligodendrocyte-Like Cells for potential clinical treatments whichrequire transplantation of neural stem cells, neurons, astrocytes oroligodendrocytes 1) to compensate for a loss of host cells (ex. neurons)or 2) as vehicles to deliver genetically-based drugs. Further, theinvention provides a novel neurological tool for use in basic researchand drug screening.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents are considered to be within the scope of this inventionand covered by the claims appended hereto. The invention is furtherillustrated by the following examples, which should not be construed asfurther limiting.

EXAMPLES

The examples set forth herein below provide exemplary methods forobtaining Reprogrammed and Dedifferentiated cells, including NeuralStem-Like Cells (NSLCs). Also provided are exemplary protocols,molecular tools, probes, primers and techniques.

Example I Preparation of Human Fibroblast Cells

Human Foreskin fibroblast (HFF) cells were purchased from American TypeCulture Collection (ATCC, Manassas, Va.) and expanded in cell cultureflasks with Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen),supplemented with 10% heat-inactivated fetal calf serum (FCS, HycloneLaboratories), 0.1 mM non-essential amino acids, and 1.0 mM sodiumpyruvate (Invitrogen) at 37° C., 5% CO₂. The medium was changed twiceper week. Cells were trypsinized using Trypsin 0.25% for 4 minutes at37° C., followed by adding trypsin inhibitor solution, pelleting thecells by centrifugation, washing the cells once with PBS, and platingthe cells at a ratio of 1:2 onto tissue culture flasks until a suitablenumber of cells was reached.

Cells were then trypsinized and plated (8×10⁴ cells/well) in cellculture plates pre-coated with Laminin (10 μg/ml, Sigma) in twodifferent composition of CDM medium: CDM I Medium consisting of a 3:1ratio of Dulbecco's modified Eagle medium (DMEM, high glucose (4.5 g/L)with L-glutamine and sodium pyruvate) and Ham's F-12 medium supplementedwith the following components: EGF (4.2×10⁻¹⁰M), bFGF (2.8×10⁻¹⁰M), ITS(8.6×10⁻⁵M), dexamethasone (1.0×10⁻⁷M), L-3,3′,5-triiodothyronine(2.0×10⁻¹⁰M), ethanolamine (10⁻⁴M), GlutaMAX™ (4×10⁻³M), and glutathione(3.3×10⁻⁶M), but without the presence of L-ascorbic acid.

CDM II Medium consisting of a 3:1 ratio of Dulbecco's modified Eaglemedium (DMEM, high glucose (4.5 g/L) with L-glutamine and sodiumpyruvate) and Ham's F-12 medium supplemented with the followingcomponents: EGF (2.5 ng/ml), bFGF (10 ng/ml), ethanolamine (2.03 mg/ml),insulin (10 mg/ml), Selenious acid (2.5 μg/ml), dexamethasone (19.7μg/ml), L-3,3′,5-triiodothyronine (675 ng/ml), GlutaMAX™ (4×10⁻³M), andglutathione (3.3×10⁻⁶M).

Transient Transfection of HFF by Lipofectamine Using Constructed Vectors

After two days in culture, cells were transfected with pCMV6-XL5-MBD2 (2μg) (a DNA demethylator) using lipofectamine reagent (Invitrogen) as perthe manufacturer's protocol. The DNA-lipid complex was added to cellsand incubated for 24 h at 37° C., 5% CO₂. After 24 hours of transfectionwith the DNA demethylator, the medium was changed and cells weretransfected by pCMV6-XL5-Musashi1 (2 μg, Origene) or pCMV6-XL4-Ngn2 (2μg, Origene) for 24 h. After 24 hours, the medium was changed to NeuralProgenitor Basal Medium (NPBM, Lonza) supplemented with Noggin (20ng/ml, Peprotech), EGF (20 ng/ml, Peprotech), and bFGF (20 ng/ml,Peprotech) and cultured in this Proliferation Medium. Cells wereretransfected after three days and incubated at 37° C., 5% CO₂ and 5%O₂. After 7 days in proliferation conditions, 50% of the ProliferationMedium was changed to Differentiation Medium (NbActive, BrainBits™)supplemented with Forskolin (10 μM, Tocris), all-trans-Retinoic Acid(ATRA, 5 μM, Spectrum), bFGF (20 ng/ml, Peprotech), NGF (20 ng/ml,Peprotech), and BDNF (20 ng/ml, Peprotech); medium was changed every dayby increasing the percentage of Differentiation Medium overProliferation Medium, and the cells were cultured for 20 days.

Visual observation of reprogrammed cells was performed by lightmicroscopic observation every day following transfection using brightfield at 10× magnification. Samples were collected at different timepoints (6, 12, and 20 days) to analyze neuronal gene expression andprotein levels by gene array and immunohistochemistry. Followingtransfection, reprogramming cells displayed a rapid change in cellularmorphology within 3 days post-transfection (FIG. 1). The cells were morerounded and the cell's cytoplasm retracted towards the nucleus formingcontracted cell bodies with extended cytoplasmic extensions andexhibiting neuronal perikaryal appearance at day 6 and 12, which wasmaintained until day 20. However, this morphology was not observed inuntransfected cells at day 6 and 12.

Gene Array Analysis

Characterization of the newly engineering cells after transfection wasperformed using a neuronal gene-array containing 48 partial cDNAs codingfor these genes and controls.

RNA was isolated from samples using QIAshredder™ (Qiagen) and RNeasy™Plus mini Kit (Qiagen) as per manufacturer's instructions. DNase Itreatment was performed on the RNeasy™ Column to further remove thetransfected plasmid DNA using Rnase-Free DNase Set (Qiagen). RNA waseluted in 35 μl of RNase-free water. Before cDNA synthesis, all RNAsamples were quantified using the NanoDrop 1000™ (ThermoScientific).cDNA was prepared using the High Capacity cDNA archive kit (AppliedBiosystems) as per the manufacturer's instructions. 400 ng of RNA wasused in each 50 μl RT reaction. The resulting cDNA samples were usedimmediately for TLDA analysis. For each card of the Taqman™ low-densityarray (TLDA), there are eight separate loading ports that feed into 48separate wells for a total of 384 wells per card. Each 2 μl wellcontains specific, user-defined primers and probes, capable of detectinga single gene. In this study, a customized Neuronal Markers 2 TLDA wasconfigured into eight identical 48-gene sets, i.e. 1 loading port foreach 48-gene set. Genes were chosen based on literature. Each set of 48genes also contains three housekeeping genes: ACTIN, GAPDH, and PPIA.

A sample-specific master mix was made for each sample by mixing cDNA(160 ng for each loading port), 2× Taqman™ Gene Expression Master Mix(Applied Biosystems) and nuclease-free water (USB) for a total of 100 μlper loading port. After gentle mixing and centrifugation, the mixturewas then transferred into a loading port on a TLDA card. The array wascentrifuged twice for 1 minute each at 1200 rpm to distribute thesamples from the loading port into each well. The card was then sealedand PCR amplification was performed using Applied Biosystems 7900HT™Fast Real-time PCR system. Thermal cycler conditions were as follows: 2minutes at 50° C., 10 minutes at 94.5° C., and 30 seconds at 97° C., 1minute at 59.7° C. for 40 cycles. 1 TLDA's was prepared for 8 samples.

Relative Expression values were calculated using the Comparative C_(T)method. Briefly, this technique uses the formula 2^(−ΔΔCT) to calculatethe expression of target genes normalized to a calibrator. The thresholdcycle (C_(T)) indicates the cycle number at which the amount ofamplified target reaches a fixed threshold. C_(T) values range from 0 to40 (the latter representing the default upper limit PCR cycle numberthat defines failure to detect a signal). ΔC_(T) values [ΔC_(T)=C_(T)(target gene)−C_(T) (Average of 3 Housekeeping genes)] were calculatedfor HFF Ctrl, and subsequently used as the calibrator for the respectivesamples. All gene expression values were assigned a relative value of1.00 for the calibrator, which is used to determine comparative geneexpression such that ΔΔC_(T)=ΔC_(T) (Treated)−ΔC_(T) (HFF Ctrl).Relative Expression is calculated using the formula 2^(−ΔΔCT).

Quantitative comparison of astrocyte, neuron, and oligodendrocyte geneexpression allowed identification of the majority of the genes that aredifferentially expressed in reprogrammed cells. Data in Table 1 wereanalyzed by using a significance analysis algorithm to identify genesthat were reproducibly found to be enriched in reprogrammed cellscompared to untransfected cells. After the transfection with Msi1 orNgn2 in the presence of MBD2, the expression of oligodendrocytesprogenitors such as NKx2.2, olig2, and MAG and two markers forastrocytes (GFAP and AQP4) were highly increased. Also, several markersof early neuronal cells were enhanced after the transfection of HFF.TDLA data revealed a remarkable increase in specific markers forinterneurons, such as somatostatin and calbindin1. The induction ofDoublecortin (DCX), which is expressed by migrating immature cellsduring development, and acetylcholine (ACHE) mRNA, an early marker ofneuronal cells, were highly expressed in the reprogrammed cells (Table1). Transfection increased the expression of dihydropyrimidinase-like 3(DPYSL3), an early marker of newborn neurons, to fivefold with Msi1 andseven fold with Ngn2. Expression of Microtubule-Associated Protein 2(MAP2), an essential marker for development and maintenance of earlyneuronal morphology and neuronal cell adhesion molecule, were highlyexpressed with Msi1 and Ngn2 (Table 1). The expression of enolase-2, amarker of mature neurons, was 20-fold enhanced by Msi1 and Ngn2. Memberof the NeuroD family NeuroD1 was highly expressed after transfectionwith Msi1 to 84 fold and to 34 by Ngn2.

Gene expression of growth factors such as IGF-1, IGF2, NPY and CSF-3 wasalso enhanced in reprogrammed cells. The expression of VEGF and GDNFgenes were up-regulated to almost five fold and seven fold by Msi1 andNgn2, respectively. However, the expression of BDNF, EGF, and bFGF werenot activated and even down-regulated as compared to untransfectedcells. The expression of growth associated protein (GAP-43), a growth-and regeneration-associated marker of neurons, and expression of netrin,implicated in neuronal development and guidance, were highly enriched inreprogrammed cells. Expression of receptors for growth and neurotrophinfactors was increased, such as type III receptor tyrosine kinase,neurotrophic tyrosine kinase, and neurotrophic tyrosine kinase receptor.Vimentin and fibronectin, markers for fibroblasts, were down-regulatedin reprogrammed cells compared to the untransfected control fibroblastcultures.

TABLE 1 Gene array of transfected human fibroblast cells by Msi1/MBD2and Ngn2/MBD2. Gene array was performed on samples after two weeks ofdifferentiation. Expression values are given relative to untransfectedfibroblasts. Relative Relative Company expression expression SymbolCommon name and description Gene ID to Msi1 to Ngn2 Astrocytes andoligodendrocytes markers NKx2-2 Markers for oligodendrocyte progenitorsNM_002509.2 very high very high OLIG2 Oligodendrocyte lineagetranscription factor 2 NM_005806.2 47.511 8.38 MAG Myelin-associatedglycoprotein NM-080600.1 212.61  4.51 GFAP Glial fibrillary acidicprotein NM_002055.4 very high very high AQP4 Aquaporin 4 NM_001650.483.77  56.86  NC markers SST Somatostatin, specific marker forinterneurons NM_001048.3 32.73  35.34  CALB1 Calbindin 1, interneuronmarker NM_004929.2 18.21  13.22  Tubulin1A Are necessary for axonalgrowth NM_006009.2 7.45 9.32 NES Precursor neurons (nestin) NM_006617.11.61 1.54 DCX An early neuronal marker (Doublecortin) NM_178151.1 veryhigh very high ACHE Acetylcholinesterase, marker of early neuronalNM_015831.2 9.02 13.22  development ENO2 A marker for neurons cells,enolase NM_001975.2 22.62  20.68  NEUROD1 Neural marker; expressiongradually increased NM_002500.2 84.22  34.27  from neural precursor tofully differentiated neuron DPYSL3 Dihydropyrimidinase-like3, marker ofNM_001387.2 5.33 7.02 immature neurons MAP2 Microtubule-associatedprotein 2, essential for NM_002374.3 86.38  89.67  development of earlyneuronal morphology and maintenance of adult neuronal morphology NCAMNeural cell adhesion molecule 1 NM_18135.3 very high very high CEND1Cell cycle exit & neuronal differentiation, early NM_016564.3 4.80 5.57marker of proliferating precursor cells that will differentiate toneurons Neuroregeneration and survival genes FGF2 Fibroblast growthfactor NM_002006.4 0.06 0.11 EGF Epidermal growth factor, Hs00153181_m10.99 0.56 IGF-1 Insulin growth factor-1, NM_000618.2 58.92  21.21  IGF-2Insulin growth factor-2 NM_0000612.3 very high very high CSF3Granulocyte colony-stimulating factor NM_2219.1 very high 42.60  BDNFBrain derived growth factor, neurogenesis NM-199231.1 0.05 0.03 GDNFGlial derived neurotrophic factor NM-000614.2 4.77 6.89 CNTF Ciliaryneurotrophic factor NM_001025366.1 1.86 1.09 VEGF Vascular endothelialgrowth factor 6.67 7.32 BMP-4 Bone morphogenetic protein 4 NM_130850.15.96 8.57 KDR Type III receptor tyrosine kinase) NM_002253.1 31.78  6.83NTRK2 Neurotrophic tyrosine kinase receptor (TrkB) NM_006180.3 10.31 13.37  NPY Neuropeptide factors NM_00905.2 very high very high PIK3CGphosphoinositide-3-kinase, NM_002649.2 very high very high STAT3 Signaltransduction transcription 3 NM_213662.1 2.14 3.65 Gap43 Growthassociated protein 43 NM_002045.2 very high very high NTN1 Netrin1,implicated in neuronal development _NM_006180.3 26.84  23.98  andguidance NTRk2 Neurotrophic tyrosine kinase, receptor, type 2NM_024003.1 10.31  13.37  Slit Axonal guidance molecules NM_003061.1very high very high Vimentin Radial glia and fibroblast markerHs00185584 0.11 0.13 Fibronectin fibronectin is a marker for fibroblastsNM_212474.1 0.15 0.23

Immunohistochemical Analysis

Cells were fixed with a 4% formaldehyde/PBS solution for 10 min at roomtemperature and subsequently permeabilized for 5 min with 0.1% TritonX-100™ in 4% formaldehyde/PBS. After two brief washes with PBS,unspecific antibody binding was blocked by a 30 min incubation with 5%normal goat serum in PBS. Then primary antibodies were added in 5%normal goat serum/PBS as follows: Mouse anti-Nestin (1:100, BD) as anintermediate microfilament present in neural stem cells and mouseanti-NCAM (1:100, Neuromics) as neuronal adhesion molecule. After a 2 hincubation the cells were washed 4 times for 5 min each with 0.1%Tween™/PBS. Appropriate fluorescence-tagged secondary antibody was usedfor visualization; Goat anti-mouse 546 (1:200, invitrogen) prepared in5% normal goat serum/PBS was used. After incubation for one hour, cellswere washed in 0.1% Tween™/PBS three times for 5 min each. The DNA stainHoechst33342 (Invitrogen) was used as a marker of nuclei (dilution1:5000 in PBS, 10 min incubation). Fluorescence images were taken with aCellomics™ ArrayScan HCS Reader microscopy system. To determine anestimate of the percentage of cells adopting neuronal or glialphenotypes, random fields were selected and for each field the totalnumber of cells (as determined by counting Hoechst stained nuclei) andthe total number of cells positive for neuronal or glial markers weredetermined.

To confirm that these cells exhibited markers of neuronal lineages,cells were immunostained for nestin and NCAM. This analysis revealedthat reprogrammed cells expressed both proteins. As shown in FIG. 2,NCAM was present in cells during the 6 days post-transfection andincrease at day 12 and 20 following differentiation, while the inversepattern was observed for the nestin staining.

This study showed the ability to reprogram HFF cells using oneneurogenic transcription factor with the presence of a DNA demethylatortowards cells that expressed neuronal genes and proteins specific toneural stem cells and neuronal cells. These reprogrammed cells werestable in culture for at least 2 weeks.

Example II Comparison of Reprogramming Efficiency of Three DifferentNeurogenic Genes

HFF cells were cultured as described in Example I and plated in CDM Imedium. Cells were transfected using the Amaxa Nucleofector™ Device(Lonza). The HFFs were harvested with TrypLE™ (Gibco), resuspended inCDM Medium and centrifuged for 10 min at 90×g (1×10⁶ cells/tube). Thesupernatant was discarded and gently resuspended in 100 μl of BasicNucleofector™ Solution (basic Nucleofector™ kit for primary mammalianfibroblasts, Lonza). Each 100 μl of cell suspension was combined with adifferent mix of plasmid DNA (for example, sample 1 was mixed with 2 μgof pCMV6-XL5-Pax6 and 2 μg pCMV6-XL5-MBD2). Cell suspension wastransferred into an Amaxa certified cuvette and transfected with theappropriate program (U023). The sample was transferred without anyfurther resuspension into a coated culture plate with LAS-Lysine/Alanine(BrainBits™, 50 μg/ml) and the cells were incubated at 37° C., 5% CO₂.These steps were repeated for each sample that was transfected. After 24hours, the medium was changed to Proliferation Medium. After two days,cells were retransfected using lipofectamine as described in Example Iand incubated at 37° C., 5% CO₂ and 5% O₂. After 6 days, differentiationwas induced with Differentiation Medium that gradually replaced theProliferation Medium over several days. Cells were collected at day 14for RT-PCR and immunohistochemistry analysis.

Gene Expression Analysis

RNA isolation and quantification was performed as previously describedin Example I. cDNA was prepared using the High Capacity cDNA RT kit(Applied Biosystems) as per the manufacturer's instructions with a finalcDNA concentration of 2 ng/μl. Real-time PCR was then performed for eachgene of interest using the FAST PCR master mix (Applied Biosystems) andthe Taqman™® Gene Expression Assays (Applied Biosystems) listed below:

Gene Name Assay ID ACHE Hs00241307_m1 NES Hs00707120_s1 TUBB3Hs00964962_g1 GFAP Hs00157674_m1 PAX6 Hs00240871_m1 MSI1 Hs01045894_m1NGN2 Hs00702774_s1 MAP2 Hs00258900_m1 GAPDH (housekeeping gene)Hs99999905_m1 PPIA (housekeeping gene) Hs99999904_m1

The FAST 96-well reaction was performed with 8 ng cDNA per well in a 10μl reaction with 40 cycles. Thermal cycler conditions were as follows:20 seconds at 95° C., and 1 second at 95° C., 20 seconds at 60° C. for40 cycles.

Relative Expression values were calculated as previously described inExample I, except the Average of 2 Housekeeping genes (GAPDH & PPIA) wasused for normalization instead of the Average of 3 Housekeeping genes.Identification of neuronal lineage genes was investigated following thetransfection with three independent vectors containing Msi1, Ngn2, andPax6.

As shown in Table 2, after 14 days following transfection, relativeexpression of mRNA of neuronal lineage was undetectable in untransfectedcells (HFF), while the cells transfected with Msi1 or Ngn2 in thepresence of MBD2 expressed neural stem cell markers (Nestin and Sox2),however the expression of Sox2 was much more highly expressed thannestin following transfection with Ngn2 or Msi1. Neuronal and astrocytespecific genes (βIII-Tubulin, MAP2b, GFAP, and ACHE) was increased aswell. mRNAs level of the tripotent-associated genes βIII-tubulin, MAP2b,acetycholine, and GFAP were undetectable in Pax6 transfected cells,indicating that Pax6 alone was not implicated in the reprogrammingprocess toward neuronal lineage.

TABLE 2 Relative expression of gene expression of different neuronallineage performed by RT-PCR following the transfection of HFF by Msi1,Ngn2, or Pax6 in the presence of MBD2 and cultured for 14 days. MSI1NGN2 PAX6 NES TUBB3 Rel. Std. Std. Std. Rel. Std. Rel. Std. Exp. Dev.Rel. Exp. Dev. Rel. Exp. Dev. Exp. Dev. Exp. Dev. #1 Control 1.00 0.071.08 0.57 1.11 0.67 1.00 0.02 1.00 0.01 Untransfect. #2 4077.82 248.021.18 0.66 487.09 69.58 8.62 0.00 6.58 0.11 MSI1/MBD2 #3 14.16 0.6347803.26 192.78 624.31 91.27 8.62 0.02 8.33 0.02 NGN2/MBD2 #4 1.70 0.360.27 0.01 29564.43 357.89 0.46 0.00 0.49 0.02 PAX6/MBD2 ACHE GFAP MAP2SOX2 Rel. Std. Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp.Dev. Exp. Dev. #1 Control 1.02 0.29 1.00 0.06 1.00 0.01 1.00 0.09Untransfect. #2 6.58 0.64 215.71 20.65 5.50 0.46 3499.53 184.85MSI1/MBD2 #3 8.33 0.97 365.60 5.11 5.42 0.00 4039.03 8.65 NGN2/MBD2 #41.98 0.48 1.15 0.13 0.55 0.04 1.00 0.03 PAX6/MBD2

Immunohistochemical Analysis

Fluorescent immunohistochemical staining was performed as previouslydescribed in Example I. In agreement with the RT-PCR data,immunohistochemical analysis of these cultures revealed thatreprogrammed cells (with Msi1 or Ngn2) generated morphologically complexneurons that were positive for MAP2b, indicating the differentiation ofNSLCs to neuron-like cells (NLCs) (FIG. 3). However, the positivestaining for these markers was undetectable after transfection withPax6/MBD2. Moreover, the newly formed neurons expressed the markers forand developed long neurites with growth cones at their ends, expressedneural specific genes, and ceased to proliferate when they were exposedto differentiation conditions.

Example III Transfection of HFF by Various Combinations of Vectors andDisruption of Cell Cytoskeleton

Various combinations of neurogenic regulators and cytokines forepigenetic modifications were tested to ascertain their effect onreprogramming efficiency. Starting one day before transfection, cellswere treated with or without cytochalasin B (Calbiochem), with theconcentration decreased every day over five days during media changes(starting with 10 μg/ml Cytochalasin B on day 1 to 7.5 μg/ml, 5 μg/ml,2.5 μg/ml, and 0 μg/ml over the subsequent four days) in order toinvestigate the effect of disrupting the cell cytoskeleton on theprocess of reprogramming. Cells were transiently transfected asdescribed in Example II with one or two vectors containing oneneurogenic transcription factors by nucleofection. Cells wereco-transfected with either of two DNA demethylators, MBD2 or GAdd45B,(e.g. 2×10⁶ cells were transfected with pCMV6-XL5Msi1 (2 μg) andpCMV6-XL5-MBD2 (2 μg)). After 24 hours, the medium was changed to NeuralProliferation Medium (NeuroCult™ proliferation Kit, StemCellTechnologies) consisting of DMEM/F12 (1:1), glucose (0.6%), sodiumbicarbonate (0.1%), glutamine (20 mM), HEPES (5 mM), insulin (230μg/ml), transferrin (100 μg/ml), progesterone (200 nM), putrescine (90μg/ml), and sodium selenite (300 nM) and supplemented with Noggin (20ng/ml, Peprotech), recombinant hFGF (20 ng/ml, Peprotech), andrecombinant hEGF (20 ng/ml, Peprotech) and cells were cultured for twoweeks at 37° C., 5% CO₂ and 5% O₂. Cells were then analyzed for neuralstem cell markers.

Gene Expression Analysis

Gene expression analysis was performed for neural stem-specific markers(Sox2, Nestin, GFAP) and a fibroblast-specific marker (Col5A2) by RT-PCRas previously described in Example I. RT-PCR analysis showed that therelative expression of Sox2, nestin and GFAP was enhanced aftertransfecting the cells with the neurogenic transcription factors. Asshown in Table 3, transfecting the cells with one transcription factorMsi1 in the presence of Gadd45b was associated with up-regulation ofrelative expression of Sox2 (22.3±5.26) and GFAP (10.14±0.15) and theexpression of the these genes was highly increase when transfecting thecells with Ngn2 by 20 fold and 10 fold respectively. Combining the twoneurogenic factors (Msi1 and Ngn2) with Gadd45b enhanced further theexpression of Sox2 and GFAP. Transfecting the cells with onetranscription factor (Msi1 or Ngn2) in the presence of MBD2 wasassociated with up-regulation of relative expression of Sox2, Nestin,and GFAP and down-regulation of Col5A2, while co-transfection withGadd45b did not increased the expression of nestin and the expression ofCol5A2 was not regulated. The enhancement of neural stem cells relativeexpression was observed when transfecting the cells with two neurogenicgenes in combination with MBD2; a small increase in the expression wasnoticed in the presence of cytochalasin B under certain conditions. Anincrease in the relative expression of the neural stem-specific markers(Sox2, Nestin, GFAP) and a decrease in the fibroblast-specific gene(COL5A2) was observed after transfection with Msi1/Ngn2/MBD2,Msi1/Ngn2/Gadd45b, Msi1/MBD2 or Ngn2/MBD2 (Table 3). This studydemonstrated that MBD2 increased more reprogramming efficiency thenGDA45b and showed that cytochalasin B had no effect of its own in thecontrol cultures.

TABLE 3 RT-PCR analysis of relative expression of neuronal precursorcell markers such as nestin, Sox2, and GFAP after transfection offibroblast cells with different combinations with or without theco-treatment with cytochalasin B. Relative expression of Sox2, nestin,and GFAP in NSLCs was increased after transfection with bothtranscription factors (Ngn2 and Msi1) with MBD2 as the DNA demethyaltor.As demonstrated, this upregulation of neural stem cell gene expressionwas associated with a decrease of CoL5A2, a specific gene for fibroblastcells. COL5A2 FBN2 NES MAP2 TUBB3 SOX2 ACHE GFAP Rel. Std. Rel. Std.Rel. Std. Rel. Std. Rel. Std. Std. Rel. Std. Rel. Std. Exp. Dev. Exp.Dev. Exp. Dev. Exp. Dev. Exp. Dev. Rel. Exp. Dev. Exp. Dev. Exp. Dev.#1, +CytoB, 1.00 0.07 1.00 0.01 1.00 0.04 1.00 0.05 1.00 0.05 1.00 0.051.00 0.10 1.00 0.11 Control #2, −CytoB, 1.00 0.03 1.00 0.08 1.00 0.001.00 0.09 1.00 0.09 1.15 0.80 1.01 0.18 1.00 0.01 Control #3, +CytoB,0.85 0.04 0.75 0.02 0.60 0.01 0.29 0.01 0.44 0.00 22.39 5.26 0.81 0.1910.14 0.15 Msi1, GAD45b #4, −CytoB, 0.87 0.03 1.81 0.09 1.84 0.04 2.310.00 2.09 0.03 20.28 5.33 1.99 0.74 6.03 0.05 Msi1, GAD45b #5, +CytoB,0.84 0.04 0.77 0.03 0.44 0.00 0.24 0.00 0.36 0.01 470.84 13.43 0.63 0.05103.22 0.80 Ngn2, GAD45b #6, −CytoB, 0.75 0.07 1.97 0.02 1.83 0.00 4.400.16 2.02 0.10 789.33 60.35 1.70 0.13 110.48 4.90 Ngn2, GAD45b #7,+CytoB, 0.74 0.12 1.08 0.00 0.89 0.01 0.51 0.00 0.63 0.04 1.64 0.98 0.860.12 2.49 0.21 Pax6, GAD45b #8, −CytoB, 0.66 0.04 2.41 0.09 2.70 0.034.96 0.30 3.48 0.07 0.46 0.33 2.97 1.04 0.43 0.09 Pax6, GAD45b #9,+CytoB, 0.14 0.01 0.28 0.01 1.30 0.03 4.07 0.11 0.84 0.00 54768.276709.56 0.81 0.24 3391.96 64.63 Msi1, Ngn2, GAD45b #10, −CytoB, 0.120.00 0.73 0.03 5.28 0.21 50.84 1.23 4.93 0.28 17400.66 822.88 3.58 0.101255.76 5.27 Msi1, Ngn2 GAD45b #11, +CytoB, 0.10 0.00 0.26 0.01 1.110.01 3.69 0.09 0.76 0.00 55588.41 1331.20 0.55 0.14 2849.96 261.51 Msi1,Ngn2 MBD2 #12, −CytoB, 0.44 0.01 1.47 0.06 5.49 0.14 47.30 0.11 5.500.31 14587.46 789.19 3.90 0.13 1424.04 39.29 Msi1, Ngn2 MBD2 #13,+CytoB, 1.11 0.04 1.09 0.06 0.92 0.08 0.68 0.01 0.82 0.03 63.93 2.811.19 0.17 17.43 1.86 GAD45b #14, −CytoB, 0.94 0.01 2.22 0.00 2.82 0.026.49 0.30 4.01 0.05 6.12 0.61 2.34 0.17 1.42 0.10 GAD45b #15, +CytoB,0.83 0.00 0.83 0.05 0.36 0.01 0.16 0.01 0.36 0.00 3.42 3.74 0.63 0.372.18 0.12 MBD2 #16, −CytoB, 0.68 0.02 1.55 0.04 1.57 0.05 1.47 0.01 2.000.00 0.52 0.29 1.45 0.15 0.55 0.04 MBD2 #17, +CytoB, 1.10 0.01 1.16 0.031.37 0.01 1.12 0.06 0.86 0.06 5.59 1.48 1.07 0.27 1.70 0.46 Msi1, Ngn2#18, −CytoB, 0.93 0.04 2.52 0.10 3.48 0.01 9.01 0.02 4.55 0.18 1.78 1.463.83 0.42 0.59 0.01 Msi1, Ngn2 #19, +CytoB, 0.20 0.03 0.36 0.01 1.250.05 6.68 0.31 0.72 0.02 66592.29 3481.89 2.57 0.03 4450.08 131.85 Msi1,MBD2 #20, −CytoB, 0.12 0.00 0.64 0.03 4.70 0.22 77.51 0.11 4.12 0.1119128.03 1542.00 8.14 0.13 999.22 24.75 Msi1, MBD2 #21, +CytoB, 0.170.01 0.28 0.00 1.16 0.04 5.73 0.06 0.62 0.00 67945.51 3000.74 2.15 0.044736.83 11.92 Ngn2, MBD2 #22, −CytoB, 0.17 0.00 0.78 0.03 4.32 0.0868.89 5.26 4.01 0.04 16570.91 92.96 7.04 0.53 1427.13 13.19 Ngn2, MBD2#23, +CytoB, 0.71 0.05 0.79 0.06 0.87 0.01 0.63 0.06 0.67 0.04 2.86 0.701.08 0.08 2.08 0.11 Msi1 #24, −CytoB, 0.66 0.04 1.92 0.17 2.03 0.02 2.770.02 2.68 0.02 0.32 0.12 1.85 0.65 0.58 0.04 Msi1

Immunohistochemical Analysis

Fluorescent immunohistochemical staining was performed as previouslydescribed in Example I. Table 4 shows the percentage of Nestin and Sox2in each condition, with the highest percentage of Sox2 (38.18±1.75%) andnestin (28.18±2.77%) positive cells observed after transfecting thecells simultaneously with both neurogenic transcription factors and inthe presence of a DNA demethylator and cytochalasin B. A slight increaseof Sox2 positive cells (10.42±10.27%) and nestin positive cells(4.85±1.10%) was detected following transfection with one transcriptionfactor Msi1 and MBD2. Same tendency of nestin and Sox2 positive cellswas observed following transfection with Ngn2 and MBD2. Disrupting thecell cytoskeleton with Cytochalasin B significantly enhancedreprogramming, but had no reprogramming effect on its own (Table 4).

TABLE 4 Percentage of positive cells for Sox2 and nestin aftertransfection of fibroblast cells with different expression vectors withor without the presence of cytochalasin B. After transfection the cellswere cultured in proliferation medium (StemCell Technologies)supplemented by EGF (20 ng/ml, Peprotech) and FGF (20 ng/ml, Peprotech)for two weeks at 37° C./ 5% CO₂/5% O₂. The percentage of immunopositivecells was determined by Cellomics ™ and represented as mean ± SD (n =3-5). % of Sox2 positive cells % of Nestin positive cells +CytoB −Cyto B+CytoB −CytoB Untransfected cells 0.02 ± 0.01 0.01 ± 0.00 0.14 ± 0.040.11 ± 0.09 Ngn2 0.35 ± 0.36 0.15 ± 0.05 2.34 ± 0.99 1.04 ± 0.21 Msi10.23 ± 0.15 0.12 ± 0.09 1.95 ± 0.11 1.11 ± 0.18 Gadd45b 0.30 ± 0.17 0.29± 0.11 4.94 ± 0.25 2.33 ± 0.42 MBD2 0.22 ± 0.13 0.22 ± 0.11  2.8 ± 0.111.53 ± 0.6  Msi1/Ngn2 0.19 ± 0.13 0.32 ± 0.05 1.91 ± 0.56 2.59 ± 1.28Msi1/MBD2 10.42 ± 10.27  8.84 ± 11.63 4.85 ± 1.10 2.06 ± 0.08Msi1/Gadd45b 0.06 ± 0.01 0.14 ± 0.17 0.55 ± 0.06 0.24 ± 0.11 Ngn2/MBD211.17 ± 0.08   9.07 ± 11.31  5.7 ± 0.10 2.18 ± 0.23 Ngn2/GAdd45b 0.29 ±0.11 0.95 ± 0.17 1.17 ± 0.54 0.98 ± 0.25 Msi1/Ngn2/MBD2 38.18 ± 1.75 22.03 ± 1.90  28.18 ± 2.77  14.54 ± 0.45  Msi1/Ngn2/Gadd45b 22.65 ±5.03  18.54 ± 9.40  18.72 ± 6.26  8.70 ± 4.51

Various DNA demethylators were tested as well for their effect onreprogramming efficiency. Cells were co-transfected with one vector(MSI1/NGN2) containing two neurogenic pCMV6-Msi1-Ngn2 factors withvarious DNA demethylators. Simultaneously another neurogenic factor wastested for its effect on cells de-differentiation towards NSCs,pCMV-XL-Nestin individually or in combination with pCMV-Msi1-Ngn2,pCMV-XL5-Msi1, or pCMV-XL4-Ngn2 in the presence of MBD2 as previouslydescribed in Example II.

Cells were co-transfected pCMV-Msi1-Ngn2 with different DNAdemethylators (MBD1, MBD2, MBD3, MBD4, MeCP2, AICDA). Another assay wasperformed to assess the effect of nestin on the reprogrammingefficiency; therefore cells were transfected with nestin individually orin combination with one vector containing one neurogenic factor (Msi1 orNgn2) or both neurogenic factors in the presence of MBD2. Cells werecultured following transfection in the presence of proliferation mediumsupplemented with EGF (20 ng/ml), FGF (20 ng/ml), and Noggin (20 ng/ml)with and without VPA (1 mM) treatment for 12 days at 37° C., 5% CO₂ and5% O₂.

Gene expression analysis and immunohistochemistry was performed toanalyse neural specific gene and protein expression (βIII-tubulin, GFAP,Sox2, Nestin) as described in Example II. Transfecting cells with Msi1and Ngn2 in the presence of various DNA demethylators revealed andconfirm previous data showing that the among various DNA demethylatorsused in this study, MBD2 promotes the expression of neural stem genes(Sox2, GFAP, Nestin) as shown in Table 5. Furthermore, transfectingcells with nestin with and without the presence of one neurogenic factorhad no effect on the reprogramming efficiency into neural stem-likecells. However co-transfection with nestin and Msi1/Ngn2/MBD2 enhancedthe expression of neural stem cells genes and this increase was morepronounced in the presence of VPA.

TABLE 5 RT-PCR analysis of relative expression of neuronal precursorcell markers such as nestin, Sox2, βIII-tubulin, and GFAP aftertransfection of fibroblast cells with various combinations ofpCMV-Msi1-Ngn2 (MSI1/NGN2), pCMV-XL5-Msi1, pCMV-XL4-Ngn2, pCMV-XL-Nestin with different combinations of DNA demethylators, with andwithout the co-treatment with VPA. TUBB3 GFAP SOX2 NES Rel. Std. Rel.Std. Std. Rel. Std. Exp. Dev. Exp. Dev. Rel. Exp. Dev. Exp. Dev. Day 12,Untransfected (−VPA) 1.00 0.11 1.00 0.05 1.01 0.16 1.00 0.13 Day 12,Untransfected 1.00 0.03 1.00 0.06 1.00 0.00 1.00 0.02 (+VPA) Day 12,0.96 0.06 2.69 0.13 1.15 0.49 0.46 0.02 MSI1/NGN2/MBD1 (−VPA) Day 12,1.10 0.06 2.22 0.06 0.80 0.01 0.84 0.02 MSI1/NGN2/MBD1 (+VPA) Day 12,123.52 0.06 1638.53 99.86 61467.29 1487.21 31.77 0.17 MSI1/NGN2/MBD2(−VPA) Day 12, 232.00 0.08 1889.30 42.39 72022.15 7894.41 42.69 0.14MSI1/NGN2/MBD2 (+VPA) Day 12, 0.92 0.07 3.98 0.59 28.05 4.67 0.56 0.01MSI1/NGN2/MBD3 (−VPA) Day 12, 1.23 0.05 1.66 0.18 11.31 2.35 0.87 0.02MSI1/NGN2/MBD3 (+VPA) Day 12, 0.85 0.01 4.80 0.23 5.42 5.20 0.62 0.00MSI1/NGN2/MBD4 (−VPA) Day 12, 0.95 0.01 1.57 0.16 2.27 0.04 0.79 0.03MSI1/NGN2/MBD4 (+VPA) Day 12, 1.11 0.06 3.80 0.38 6.54 6.45 0.69 0.01MSI1/NGN2/MeCP2 (−VPA) Day 12, 1.37 0.09 1.63 0.45 10.53 10.49 1.07 0.01MSI1/NGN2/MeCP2 (+VPA) Day 12, 1.07 0.04 4.59 0.02 0.65 0.01 0.74 0.02MSI1/NGN2/AICDA (−VPA) Day 12, 1.10 0.01 2.37 0.29 1.21 0.16 0.91 0.04MSI1/NGN2/AICDA (+VPA) Day 12, Msi1/MBD2 (−VPA) 1.31 0.17 3.78 0.49 0.700.02 0.78 0.00 Day 12, Msi1/MBD2 1.36 0.07 1.75 0.31 1.26 0.03 1.15 0.03(+VPA) Day 12, Ngn2/MBD2 (−VPA) 0.85 0.06 2.93 0.51 0.79 0.05 0.58 0.02Day 12, Ngn2/MBD2 1.41 0.05 1.60 0.11 2.30 0.06 1.03 0.03 (+VPA) Day 12,Nes/Msi1 (−VPA) 0.84 0.03 3.21 0.72 0.76 0.01 0.51 0.01 Day 12, Nes/Msi1(+VPA) 0.86 0.09 1.82 0.30 2.14 1.02 0.94 0.01 Day 12, Nes/Ngn2 (−VPA)0.69 0.05 2.88 0.32 0.99 0.10 0.57 0.02 Day 12, Nes/Ngn2 (+VPA) 0.880.01 1.53 0.19 2.71 0.02 0.83 0.03 Day 12, 111.58 0.04 1423.56 82.8772069.27 624.51 51.52 0.12 Nes/MSI1/NGN2/MBD2 (−VPA) Day 12, 321.00 0.042600.14 1.90 88932.00 708.72 82.74 0.18 Nes/MSI1/NGN2/MBD2 (+VPA) Day12, Nes/MSI1/NGN2 0.74 0.11 2.60 0.28 1.98 0.97 0.55 0.01 (−VPA) Day 12,Nes/MSI1/NGN2 0.86 0.00 1.70 0.49 1.70 0.04 0.88 0.05 (+VPA) Day 12,Nes/MBD2 (−VPA) 0.76 0.12 3.15 0.17 0.87 0.03 0.44 0.00 Day 12, Nes/MBD20.87 0.03 2.05 0.07 2.66 1.64 0.91 0.00 (+VPA) Day 12, Nes/Msi1/MBD2(−VPA) 0.81 0.05 3.41 0.66 1.11 0.01 0.58 0.01 Day 12, Nes/Msi1/MBD21.01 0.13 2.43 0.07 3.27 0.26 0.93 0.02 (+VPA) Day 12, Nes/Ngn2/MBD21.19 0.07 5.71 1.30 4.11 0.07 0.91 0.04 (−VPA) Day 12, Nes/Ngn2/MBD21.29 0.03 2.98 0.66 21.20 0.42 1.65 0.02 (+VPA)

Immunohistochemistry analysis performed in parallel with RT-PCR dataindicated that positive Sox2 cells were undetectable when transfectingthe cells with Msi1/Ngn2 in the presence of MBD1, MBD3, MBD4, MeCP1, orAICADA (Table 6) and that among the different types of DNA demethylatorgenes tested only MBD2 plays a significant positive role in thereprogramming efficiency of HFF towards NSLCs when using the aboveneurogenic genes. Immunohistochemistry analysis revealed a smallincrease of immunopositive Sox2 cells (89.49±3.18) after co-transfectingthe cells with nestin and Msi1/Ngn2 in the presence of MBD2 (Table 6).

TABLE 6 Percentage of positive cells for Sox2 after transfection offibroblast cells with different expression vectors with or without thepresence of various DNA demethylators. After transfection the cells werecultured in proliferation medium (StemCell Technologies) supplemented byEGF (20 ng/ml, Peprotech) and FGF (20 ng/ml, Peprotech) for two weeks at37° C./5% CO₂/5% O₂. The percentage of immunopositive cells wasdetermined by Cellomics ™ and represented as mean ± SD (n = 3-5). % Sox2positive ± stdv HFF untransfected 0.13 ± 0.12 Msi-Ngn2 + MBD1 0.92 ±0.13 Msi-Ngn2 + MBD2 79.44 ± 9.86  Msi-Ngn2 + MBD3 1.22 ± 0.82Msi-Ngn2 + MBD4 0.59 ± 0.03 Msi-Ngn2 + MeCP2 1.10 ± 0.25 Msi-Ngn2 +AICDA 0.69 ± 0.28 Msi + MBD2 0.79 ± 0.28 Ngn2 + MBD2 1.74 ± 1.01Nestin + Msi 0.91 ± 0.01 Nestin + Ngn2 2.16 ± 1.44 Nestin + MSI1/NGN2 +MBD2 89.49 ± 3.18  Nestin + MSI1/NGN2 10.20 ± 0.21  Nestin + MBD2 0.00 ±0.00 Nestin + Msi + MBD2 8.45 ± 0.08 Nestin + Ngn2 + MBD2 5.71 ± 0.66

Another study was designed to test the effect of various neurogenicgenes on the reprogramming efficiency towards neural stem-like cells.HFF cells were cultured as described in Example I, and transfected usingthe Nucleofector™® 96-well Shuttle® Device (Lonza) following proceduredescribed in Example IV, except for the untreated HFF control and theuntransfected HFF control (for determining the effect of the completemedia & compound treatments on the cells). The cells that had beenpre-treated with VPA and 5-Aza and the untreated cells were transfectedwith the mixes of DNA as described in Table 7. The cells were plated onLaminin-coated plates and incubated at 37° C., 5% CO₂. Media was changeddaily according to Table 7. Cells were analysed at day 3, 7, 12 byimmunohistochemistry analysis and at Day 9 by gene array for multipotentand pluripotent gene expression.

Gene Array Analysis

An additional batch of cells treated according to 0a and 1a in Table 7was analyzed at Day 9, along with HFFs, hNPCs, and passage 5 NSLCs(frozen from previous experiments from Example III) by the PluripotencyGene Array (ABI) (Tables 8a and b) and a set of genes (Table 8c) todetermine the gene expression profile of select pluripotency, ectoderm,endoderm, mesoderm, and neural lineage genes in passage 1 and passage 5NSLCs compared to HFFs (from which they were created) and normal humanneuroprogenitor cells (hNPCs). The results in Table 8 indicate that allthe genes related to neural stem cells (some of the significantlyexpressed pluripotency markers and mesendoderm markers are alsoexpressed in neural stem cells) and the neural lineage weresignificantly expressed in NSLCs as opposed to HFFs, and the expressionpattern was a bit different from hNPCs indicating that NSLCs are similarto, but not identical, to the hNPCs tested. Passage 5 NSLCs 5 had ahigher expression of stemness genes than Passage 1 NSLCs. hNPCs had ahigher expression of neuronally committed genes than NSLCs, indictingtheir neuroprogenitor status versus the greater stemness status ofNSLCs.

TABLE 7 Plasmids and media composition from day 1 to day 12. From day −2to day 0 Plasmids transfected at day 0 From day 1 to day 3 From day 3 today 4 From day 4 to day 12  0a Untreated No plasmid Neural proliferationNeural proliferation Neural proliferation medium + Egf + Fgf-2 medium +Egf + Fgf-2 medium + Egf + Fgf-2  1a Untreated Msi1/Ngn2 + pCMV6- Neuralproliferation Neural proliferation Neural proliferation XL5-MBD2medium + Egf + Fgf-2 medium + Egf + Fgf-2 medium + Egf + Fgf-2  1bUntreated Msi1/Ngn2 + pCMV6- Neural proliferation Neural proliferationNeural proliferation XL5-MBD2 medium + Egf + Fgf-2 medium + Egf + Fgf-2medium + Egf + Fgf-2 + SHH  1c Untreated Msi1/Ngn2 + pCMV6- Neuralproliferation Neural proliferation Neural proliferation XL5-MBD2medium + Egf + Fgf-2 + medium + Egf + Fgf-2 + medium + Egf + Fgf-2 +Noggin Noggin Noggin (day 1 to day 7)/ Forskolin (day 7 to day 12)  1dUntreated Msi1/Ngn2 + pCMV6- Neural proliferation Neural proliferationNeural proliferation XL5-MBD2 medium + Egf + Fgf-2 medium + Egf + Fgf-2medium + Egf + Fgf-2  1e Untreated Msi1/Ngn2 + pCMV6- Neuralproliferation Neural proliferation Neural proliferation XL5-MBD2medium + Egf + Fgf-2 medium + Egf + Fgf-2 medium + Egf + Fgf-2  1fUntreated Msi1/Ngn2 + pCMV6- Neural proliferation Neural proliferationNeural proliferation XL5-MBD2 medium + Egf + Fgf-2 medium + Egf + Fgf-2medium + Egf + Fgf-2  2 Untreated Msi1/Ngn2 + pCMV6- Neuralproliferation Neural proliferation Neural proliferation XL5-MBD2medium + Egf + Fgf-2 + medium + Egf + Fgf-2 + medium + Egf + Fgf-2 CytoBCytoB  3 Untreated Msi1/Ngn2 Neural proliferation Neural proliferationNeural proliferation medium + Egf + Fgf-2 + medium + Egf + Fgf-2medium + Egf + Fgf-2 VPA + 5-Aza  4 Untreated Msi1/Ngn2 Neuralproliferation Neural proliferation Neural proliferation medium + Egf +Fgf-2 medium + Egf + Fgf-2 medium + Egf + Fgf-2  5 UntreatedpCMV6-XL5-Musashi Neural proliferation Neural proliferation Neuralproliferation medium + Egf + Fgf-2 medium + Egf + Fgf-2 medium + Egf +Fgf-2  6 Untreated pCMV6-XL5-Musashi Neural proliferation Neuralproliferation Neural proliferation medium + Egf + Fgf-2 + medium + Egf +Fgf-2 + medium + Egf + Fgf-2 + Noggin Noggin Noggin + Forskolin  7Untreated pCMV6-XL5-Musashi Neural proliferation Neural proliferationNeural proliferation medium + Egf + Fgf-2 + medium + Egf + Fgf-2medium + Egf + Fgf-2 VPA + 5-Aza  8 Untreated pCMV6-XL5-Musashi Neuralproliferation Neural proliferation Neural proliferation medium + Egf +Fgf-2 + medium + Egf + Fgf-2 + medium + Egf + Fgf-2 + Noggin + VPA +5-Aza Noggin Noggin + Forskolin  9 Untreated pCMV6-XL5-ZIC1 + Neuralproliferation Neural proliferation Neural proliferation pCMV6-XL4-Ngn2 +medium + Egf + Fgf-2 medium + Egf + Fgf-2 medium + Egf + Fgf-2pCMV6-XL5-MBD2 10 Untreated pCMV6-XL5-SOX1 + Neural proliferation Neuralproliferation Neural proliferation pCMV6-XL4-Ngn2 + medium + Egf + Fgf-2medium + Egf + Fgf-2 medium + Egf + Fgf-2 pCMV6-XL5-MBD2 11 UntreatedpCMV6-XL5-Sox2 + Neural proliferation Neural proliferation Neuralproliferation pCMV6-XL4-Ngn2 + medium + Egf + Fgf-2 medium + Egf + Fgf-2medium + Egf + Fgf-2 pCMV6-XL5-MBD2 12 Untreated pCMV6-XL5-Nanog +Neural proliferation Neural proliferation Neural proliferationpCMV6-XL4-Ngn2 + medium + Egf + Fgf-2 medium + Egf + Fgf-2 medium +Egf + Fgf-2 pCMV6-XL5-MBD2 13 Untreated pCMV6-XL4-Oct4 + Neuralproliferation Neural proliferation Neural proliferation pCMV6-XL4-Ngn2 +medium + Egf + Fgf-2 medium + Egf + Fgf-2 medium + Egf + Fgf-2pCMV6-XL5-MBD2 14 VPA + 5-Aza Msi1/Ngn2 Neural proliferation Neuralproliferation Neural proliferation pre-treated medium + Egf + Fgf-2medium + Egf + Fgf-2 medium + Egf + Fgf-2 15 VPA + 5-AzapCMV6-XL5-Musashi Neural proliferation Neural proliferation Neuralproliferation pre-treated medium + Egf + Fgf-2 + medium + Egf + Fgf-2medium + Egf + Fgf-2 VPA + 5-Aza 16 VPA + 5-Aza pCMV6-XL5-Musashi Neuralproliferation Neural proliferation Neural proliferation pre-treatedmedium + Egf + Fgf-2 + medium + Egf + Fgf-2 + medium + Egf + Fgf-2 +Noggin + VPA + 5-Aza Noggin Noggin + Forskolin 17 VPA + 5-AzapCMV6-XL5-Musashi + Neural proliferation Neural proliferation Neuralproliferation pre-treated pCMV6-XL5-MBD2 medium + Egf + Fgf-2 + medium +Egf + Fgf-2 + medium + Egf + Fgf-2 + Noggin + VPA + 5-Aza NogginNoggin + Forskolin 18 VPA + 5-Aza pCMV6-XL4-Ngn2 Neural proliferationNeural proliferation Neural proliferation pre-treated medium + Egf +Fgf-2 + medium + Egf + Fgf-2 + medium + Egf + Fgf-2 + Noggin + VPA +5-Aza Noggin Noggin + Forskolin 19 VPA + 5-Aza pCMV6-XL5-MBD2 Neuralproliferation Neural proliferation Neural proliferation pre-treatedmedium + Egf + Fgf-2 + medium + Egf + Fgf-2 + medium + Egf + Fgf-2 +Noggin + VPA + 5-Aza Noggin Noggin + Forskolin 20 VPA + 5-Aza Ngn2 +pCMV6-XL5- Neural proliferation Neural proliferation Neuralproliferation pre-treated MBD2 medium + Egf + Fgf-2 + medium + Egf +Fgf-2 + medium + Egf + Fgf-2 + Noggin + VPA + 5-Aza Noggin Noggin +Forskolin 21 VPA + 5-Aza No plasmid Neural proliferation Neuralproliferation Neural proliferation pre-treated medium + Egf + Fgf-2 +medium + Egf + Fgf-2 + medium + Egf + Fgf-2 + Noggin + VPA + 5-AzaNoggin Noggin + Forskolin * Immunohistochemistry analysis performed inparallel with RT-PCR data indicated among all the combinations in thisexperiment where no cytochalasin B was used, positive Sox2 cells weredetectable only in cells transfected with Msi1/Ngn2 with and withoutMBD2.

TABLE 8a Results for Human Stem Cell Pluripotency Array (n = 4 for eachsample) - Embryonic Stem Cell Markers, Germ Cell Markers and TrophoblastMarkers. For Relative Expression calculations, each sample wasnormalized to the average Ct of the 6 housekeeping genes (ACTB, 18S,CTNNB1, EEF1A1, GAPD, RAF1), and calibrated to the Untreated HFF(Passage 8) control. Relative Expression values with asterisk (*)indicate values with significant up or down-regulation (>2-fold or<0.5-fold). For these samples, for Ct values <35 is considered that theexpression of the gene is adequate for quantification. For the RelativeExpression values that are >2-fold or <0.5-fold but without asterisk,the values could have significant error due to the low expression of thegene (Ct > 35), and thus the up or down-regulation could be merely aresult of the high standard deviation of the high Ct values of thegenes, or fluctuations in the housekeeping genes. As for the RelativeExpression values that are between 0.5-fold and 2-fold, it indicates nosignificant change in the expression of the gene for these samples.MSI1/NGN2/MBD2- transfected HFF Neural stem-like cells, Untreated HFFUntransfected HFF hNPC neurospheres (Day 9) NSLC (Passage 8) (Day 9)(Passage 4) (NSLC, Passage 1) (Passage 5) Gene Rel. Exp. Std. Dev. Rel.Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std.Dev. Embryonic Stem cell markers BRIX 1.03 0.30 0.47 0.10 0.78 0.22 0.780.25 0.83 0.10 CD9 1.01 0.18 2.46* 0.62 1.86 0.29 2.24* 0.19 1.00 0.39COMMD3 1.08 0.53 0.94 0.36 0.94 0.40 0.98 0.40 1.05 0.59 DNMT3B 1.070.50 0.34* 0.14 2.96* 0.84 1.90 0.41 0.35 0.34 EBAF/LEFTY2 1.00 0.002.10 0.00 7.95 4.60 7.79 4.88 70.56* 26.12 FGF4 1.00 0.00 2.10 0.00 1.440.00 1.54 0.00 1.37 0.00 FOXD3 1.00 0.00 2.10 0.00 1.44 0.00 7.13 11.18222.41* 63.43 GABRB3 1.06 0.38 4.22* 0.71 66.65* 12.52 40.01* 4.54 1.620.98 GAL 1.00 0.04 9.73* 0.32 0.03* 0.01 4.25* 0.46 2.89* 0.83 GBX2 1.000.09 0.04 0.05 45.28* 4.59 90.92* 12.14 55.22* 2.36 GDF3 1.00 0.00 2.100.00 1.44 0.00 1.54 0.00 1.37 0.00 GRB7 1.02 0.24 0.30* 0.16 0.05* 0.040.29* 0.08 0.06* 0.08 IFITM1 1.01 0.17 63.96* 6.04 0.04* 0.01 21.80*4.31 3.35* 0.63 IFITM2 1.00 0.12 3.84* 0.89 0.02* 0.00 0.65 0.11 0.43*0.09 IL6ST 1.01 0.21 2.19* 0.39 0.85 0.14 1.59 0.26 0.75 0.06 IMP2 1.110.66 1.65 0.92 1.06 0.48 0.78 0.26 1.96 0.97 KIT 1.02 0.26 1.15 0.300.02* 0.00 0.31* 0.09 0.00* 0.00 LEFTB 1.61 1.15 12.28* 7.84 5.45 3.155.58 2.65 8.96* 4.12 LIFR 2.29 3.57 13.51 16.55 6.31 7.24 12.98 9.812.85 4.31 LIN28 4.69 8.62 5.25 8.88 28.38* 19.25 26.97* 8.68 32.13*14.32 NANOG 1.71 1.97 18.61 16.43 64.94* 28.32 70.87* 9.88 5.87 3.52 NOG1.03 0.27 0.18* 0.08 0.18* 0.06 0.22* 0.06 0.02* 0.00 NR5A2 2.04 2.056.85 8.80 0.38 0.00 3.89 4.36 0.36 0.00 NR6A1 1.11 0.66 1.37 0.31 5.08*0.37 2.71* 0.63 2.04* 0.17 PODXL 1.00 0.07 0.01* 0.01 0.80 0.11 2.09*0.04 6.49* 0.64 POU5F1 1.01 0.13 0.27* 0.17 0.89 0.09 0.71 0.09 0.19*0.06 PTEN 1.00 0.02 2.68* 0.29 0.87 0.04 1.07 0.12 0.80 0.14 RESET 1.010.12 1.53 0.17 0.94 0.18 1.04 0.21 1.10 0.24 SEMA3A 1.00 0.11 1.99 0.190.66 0.05 1.05 0.11 0.90 0.16 SFRP2 1.11 0.56 122.57* 14.57 3480.98*702.37 1500.84* 272.46 2.75 2.85 SOX2 1.00 0.00 2.45 0.70 127594.46*11326.91 88615.76* 15003.70 137424.37* 26622.02 TDGF1 1.41 1.28 2.920.68 6.13 1.52 5.46 1.95 2.20 1.51 TERT 1.00 0.00 2.10 0.00 10.81 18.7510.74 18.41 6506.88* 893.84 TFCP2L1 1.00 0.00 2.10 0.00 7.84 12.80 32.4910.01 1.37 0.00 UTF1 1.00 0.00 8.21 12.23 27.86 19.24 1.54 0.00 30.6825.94 XIST 1.00 0.00 2.10 0.00 24609.46* 4337.83 22637.95* 3988.10 1.370.00 ZFP42 1.24 1.06 12.38 12.58 1.41 0.78 2.01 1.85 1.76 0.93 Germ cellmarkers DDX4 1.00 0.00 2.10 0.00 1.44 0.00 5.84 8.60 19.11 20.49 SYCP31.58 1.95 11.97 8.01 11.12 3.46 15.46 11.65 2.25 2.85 Trophoblastmarkers CDX2 1.00 0.00 2.10 0.00 1.44 0.00 1.54 0.00 1.37 0.00 CGB 1.020.24 2.08* 0.74 0.15* 0.16 0.57 0.41 0.09* 0.17 EOMES 1.51 1.14 0.330.00 0.71 0.97 0.24 0.00 0.77 1.12 GCM1 2.61 2.80 0.42 0.00 3.25 5.925.68 1.44 1.47 2.38 KRT1 1.00 0.00 2.10 0.00 1.44 0.00 1.54 0.00 1.370.00

TABLE 8b Results for Human Stem Cell Pluripotency Array (n = 4 for eachsample) - Ectoderm, Endoderm and Mesoderm Markers. For RelativeExpression calculations, each sample was normalized to the average Ct ofthe 6 housekeeping genes (ACTB, 18S, CTNNB1, EEF1A1, GAPD, RAF1), andcalibrated to the Untreated HFF (Passage 8) control. Relative Expressionvalues with asterisk (*) indicate values with significant up ordown-regulation (>2-fold or <0.5-fold). For these samples, for Ct values<35 is considered that the expression of the gene is adequate forquantification. For the Relative Expression values that are >2-fold or<0.5-fold but without asterisk, the values could have significant errordue to the low expression of the gene (Ct > 35), and thus the up ordown-regulation could be merely a result of the high standard deviationof the high Ct values of the genes, or fluctuations in the housekeepinggenes. As for the Relative Expression values that are between 0.5-foldand 2-fold, it indicates no significant change in the expression of thegene for these samples. MSI1/NGN2/ MBD2- Untreated transfected HFF HFF(Day Neural stem-like (Passage Untransfected hNPC neurospheres 9) (NSLC,cells, NSLC 8) HFF (Day 9) (Passage 4) Passage 1) (Passage 5) Rel. Std.Rel. Std. Std. Std. Std. Gene Exp. Dev. Exp. Dev. Rel. Exp. Dev. Rel.Exp. Dev. Rel. Exp. Dev. Ectoderm markers CRABP2 1.04 0.35 26.14* 4.280.01* 0.01 21.11* 2.80 0.21* 0.05 FGF5 1.01 0.15 0.21* 0.07 0.00* 0.000.10* 0.02 0.00* 0.00 GFAP 1.22 0.84 9.89* 5.46 798.04* 162.37 487.99*79.84 12052.09* 2984.71 ISL1 1.01 0.12 2.19* 0.27 0.02* 0.02 0.42* 0.080.00* 0.00 NES 1.10 0.58 3.19* 0.95 6.78* 0.95 3.84* 0.19 7.47* 0.54NEUROD1 1.00 0.00 2.10 0.00 1.44 0.00 2.32 1.57 25.54 6.42 OLIG2 1.000.00 2.10 0.00 124181.50* 14735.13 80826.42* 27820.32 36172.45* 3145.67PAX6 1.11 0.48 0.06* 0.00 533.31* 120.59 326.02* 33.14 371.42* 77.50 SYP1.02 0.25 5.22* 2.10 229.40* 22.54 143.94* 17.41 16.48* 4.47 TH 1.000.00 9.52 14.86 1218.08* 186.74 217.79* 45.71 348.31* 150.50 Endodermmarkers AFP 1.00 0.00 2.10 0.00 1.44 0.00 1.54 0.00 1.37 0.00 FN1 1.000.06 1.41 0.10 0.02* 0.00 1.96 0.19 0.00* 0.00 FOXA2 1.00 0.00 150.00*55.92 1.44 0.00 1.54 0.00 1.37 0.00 GATA4 1.00 0.00 11.93 19.67 7.2211.56 9.14 12.35 1.37 0.00 GATA6 1.00 0.09 0.37* 0.17 0.00* 0.00 0.44*0.04 0.02* 0.01 GCG 1.00 0.00 7.96 11.74 1.44 0.00 33.59* 22.17 1.370.00 IAPP 1.00 0.00 2.10 0.00 1.44 0.00 1.54 0.00 1.37 0.00 INS 1.000.00 2.10 0.00 1.44 0.00 12.67 22.26 1.37 0.00 IPF1 1.00 0.00 2.10 0.001.44 0.00 1.54 0.00 1.37 0.00 LAMA1 1.00 0.11 4.42* 0.86 78.49* 6.8243.99* 2.79 46.49* 16.59 LAMB1 1.02 0.26 12.51* 2.40 0.29* 0.09 2.27*0.77 3.89* 1.12 LAMC1 1.00 0.10 2.82* 0.10 1.54 0.33 3.01* 0.94 1.310.30 NODAL 1.00 0.00 12.16 11.62 16.27 11.25 1.54 0.00 1.37 0.00 PAX41.00 0.00 6.77 9.35 1.44 0.00 1.54 0.00 1.37 0.00 PTF1A 1.00 0.00 2.100.00 1.44 0.00 1.54 0.00 1.37 0.00 SERPINA1 1.03 0.30 0.79 0.53 0.240.00 1.52 1.17 0.99 0.68 SOX17 1.00 0.00 2.10 0.00 1.44 0.00 1.35 5.631.37 0.00 SST 1.25 1.00 52.58* 10.67 0.55 0.36 48.97* 8.70 0.92 0.42 TAT1.00 0.00 2.10 0.00 255.86* 84.52 106.04* 45.87 1.37 0.00 Mesodermmarkers ACTC 1.04 0.35 0.01* 0.00 0.02* 0.01 0.05* 0.01 0.01* 0.01 CD341.67 1.69 501.85* 61.88 45.17* 27.01 113.96* 39.39 13203.40* 5385.80CDH5 1.00 0.00 4.12 4.06 16.69 8.07 32.41* 20.31 13447.65* 3220.80COL1A1 1.01 0.12 2.28* 0.41 0.00* 0.00 0.50* 0.05 0.02* 0.00 COL2A1 3.566.27 103.52* 37.78 1813.86* 236.76 873.19* 259.80 3815.72* 839.02 DES1.00 0.07 1.94 0.33 1.09 0.33 0.87 0.07 0.22* 0.08 FLT1 1.01 0.15 0.680.29 0.00 0.00 0.46* 0.05 0.00* 0.00 HBB 3.08 4.01 0.39 0.00 0.27 0.000.29 0.00 0.26 0.00 HBZ 1.14 0.63 3.53 1.32 0.25 0.22 0.61 0.63 2.881.20 HLXB9 1.00 0.00 2.10 0.00 59.80* 16.35 24.94 3.14 35.12 40.50 MYF51.77 1.87 0.69 0.00 0.47 0.00 0.51 0.00 0.45 0.00 MYOD1 1.71 2.27 1.220.00 0.83 0.00 0.89 0.00 0.80 0.00 NPPA 1.00 0.00 2.10 0.00 96.60* 76.2318.97 26.98 32.37 10.96 PECAM1 1.00 0.00 1041.24* 150.95 31.30* 24.22964.70* 200.82 7305.03* 1127.69 RUNX2 1.01 0.12 1.76 0.37 0.09* 0.020.78 0.23 1.18 0.27 T 1.00 0.00 2.10 0.00 1.44 0.00 1.54 0.00 1.37 0.00WT1 2.09 3.13 1.11 0.00 0.76 0.00 2.72 3.82 4.24 4.21

TABLE 8c Results for relative expression of Embryonic Stem Cell,Ectoderm, Endoderm/mesoderm, and neuronal markers in untransfected andtransfected HFF with Msi1/Ngn2/MBD2 calibrated to untreated HFF (passage8). Genes with asterisk (*) indicate that the Ct values of the testsamples are within the quantifiable range (Ct<35), suggesting theexpression of the gene in the test sample is adequate forquantification. For genes without asterisk, the values may be inaccuratedue to the low expression of the gene (Ct > 35) and thus the up ordown-regulation is merely a result of the high standard deviation of thehigh Ct values of the genes, or fluctuation of the housekeeping genes;the trend for these samples may be correct, but the absolute relativeexpression values may not. Expression of NGN3 and LIN28 were also testedbut these two genes were not expressed in any of the test samples (datanot shown). RT-PCR revealed a significant increase of ectoderm andneuronal markers. MSI1/NGN2/ Untreated Untrans- MBD2- HFF fectedtransfected Neural stem-like cells, (Passage HFF hNPC neurospheres HFF(Day 9) NSLC 8) (Day 9) (Passage 4) (NSLC, Passage 1) (Passage 5) Rel.Std. Rel. Std. Std. Std. Std. Gene Exp. Dev. Exp. Dev. Rel. Exp. Dev.Rel. Exp. Dev. Rel. Exp. Dev. Embryonic Stem Cell Markers OCT4* 1.040.38 7.27 0.81 6.26 0.05 6.63 0.51 3.15 0.58 OCT4 (5′UTR) 1.04 0.41 0.080.00 2.07 0.11 1.82 0.53 0.55 0.59 NANOG (5′UTR) 1.02 0.32 19.29 2.2311.27 0.89 16.73 6.86 9.94 6.32 FBXO15* 1.05 0.46 2.58 0.45 3.57 0.235.89 1.22 1.13 0.39 ALPL* 1.03 0.33 0.57 0.73 652.20 46.60 194.23 10.8213.04 4.04 SALL4* 1.02 0.25 9.20 1.35 9.76 0.62 15.84 0.92 2.35 0.55NR0B1 (DAX1)* 1.01 0.19 18.62 4.70 2.64 0.11 11.59 3.17 0.06 0.00Ectoderm Markers ZIC1* 1.01 0.24 2.01 0.25 1889.80 93.48 1158.21 80.43156.40 12.64 SOX1* 1.00 0.01 2.05 0.06 1776.83 128.63 1052.75 243.0747.98 2.12 CDH1 (E-cadherin)* 1.00 0.01 2.05 0.06 264.59 6.22 59.14 7.5718.20 3.73 p63 1.00 0.01 68.37 72.49 18.01 5.33 39.72 12.76 37.83 6.76MSX1 1.00 0.05 4.19 0.56 0.10 0.01 1.53 0.35 0.09 0.00 NOTCH1* 1.00 0.071.26 0.08 7.38 1.20 4.51 0.54 4.75 0.26 SOX2* 1.00 0.01 2.50 0.57340909.59 5659.15 194495.82 17929.15 219269.76 31399.68 SOX2 (3′UTR)*1.00 0.01 7.74 8.11 864191.09 60204.44 452684.80 26457.70 618245.017107.48 Mesoderm/Endoderm Markers CXCR4* 1.05 0.46 12.45 5.64 5048.23172.14 2763.82 30.29 3773.11 78.89 Neuronal markers MAP2* 1.01 0.17 2.980.20 155.33 9.08 88.82 6.48 27.38 0.13 TUBB3* 1.00 0.04 0.38 0.02 1.150.05 0.89 0.05 0.98 0.09 ASCL1 (MASH1)* 1.29 1.16 11.19 0.22 42618.4668.52 23554.16 1588.45 31358.79 2301.26 NGN2* 1.00 0.01 2.05 0.06 19.456.64 247883.48 16409.80 968.11 191.73 NGN2 (3′UTR)* 1.83 2.17 1.17 0.7613.39 5.10 8.45 1.75 539.02 59.72 MSI1* 1.00 0.01 263.87 70.10 100376.3681.45 479098.05 2281.62 116105.29 2745.03 MSI1 (3′UTR)* 1.01 0.20 13.612.00 3601.96 345.79 2163.87 59.84 3698.14 160.78 ACHE* 1.00 0.00 2.000.26 25.00 3.71 12.84 0.84 21.30 0.30 Glia markers CNP* 1.01 0.18 1.430.10 3.48 0.58 2.69 0.12 1.93 0.07 SOX9* 1.00 0.04 3.54 0.06 88.25 9.7141.11 2.70 26.96 0.53 Note that custom primers (5′UTR) for detectingendogenous gene expression are generally not as sensitive and/oreffective as standard primers (from the supplier's (Origene) catalog)that dtect overall gene expression (both endogenous and exogenous) of aparticular gene.

In another part of the experiment, another batch of cells that weretransfected with Msi1/Ngn2+pCMV6-XL5-MBD2 were plated on Poly-Ornithine(30 min at RT) and Laminin (1 h at RT) coated plates in CDM II medium in5 different wells. On day 1, medium in two of the wells was switched tothe same medium as in condition 1a (Table 7) until day 12. Medium waschanged daily until day 12, at which point it was switched to eitherNS-A Differentiation Medium (StemCell Technologies) or NbActive4(BrainBits™) medium that were both supplemented with BDNF (20 ng/ml),NT-3 (20 ng/ml), NGF (20 ng/ml), Retinoic acid (5 μM), Noggin (20 ng/ml)and Forskolin (10 μM). These cells showed a typical neural stem-likecell morphology by day 7, and proliferated until day 12. During theexposure to either of the two differentiation media, these NSLC changedto a more neuronal and glial phenotype as shown in the bright fieldpictures (FIG. 21), but only expressed GFAP by Day 17 (FIG. 22).

For the other three wells, on day 1 medium was switched to either NS-ADifferentiation Medium (StemCell Technologies), NbActive4 (BrainBits),or CDM II medium; these first two were supplemented with the samecytokines as previously described but with the addition of Fgf-2 (20ng/ml). On day 12, Fgf-2 was removed from the first two differentiationmedia while cells in the CDM II medium were switched to the NS-ADifferentiation Medium (StemCell Technologies) supplemented withcytokines without Fgf-2. Between day 12 and day 17, media was changedevery two to three days. During the first 12 days of culture, cells inall 3 media developed into a mix of more spindle shaped cells comparedto untransfected fibroblasts and some into cells with a NSLC morphology;upon removal of Fgf-2 cell morphology turned into a very pronouncedneuronal shape as well as glial cells with a network established betweencells as shown in the bright field pictures (FIG. 21) that expressedGFAP and βIII-tubulin by Day 17 (FIG. 22).

An additional study was designed to assess the effect of Msi1, Ngn2 andMBD2 on their endogenous proteins levels in reprogrammed cells. Cellswere transfected with the MSI1/NGN2 vector and MBD2 as previouslydescribed and cultured in proliferation condition at 37° C., 5% CO₂ and5% O₂. Samples were collected at various time points from Day 2-10 andanalyzed by RT-PCR to investigate the expression of endogenous genes andthe expression of neural stem cell and neuronal genes at different timepoints. RT-PCR revealed a gradual loss of total Msi1, Ngn2 and MBD2 geneexpression starting from Day 2 to Day 10, with the increase in MBD2expression relative to control having been almost completely lost by Day5. This decrease was associated with a significant activation ofendogenous Msi1 and Ngn2 on Day 5, with another jump in endogenous geneexpression at Day 9 (Table 9). A significant increase in Sox2 expressionwas detected at Day 4, and the expression of this ectoderm/neural stemcell/neuronal gene continued to increase with each subsequent timepoint(Table 10). GFAP (a neural stem cell and astrocyte marker) was slightlyelevated already from Day 2 onwards, but significantly increased on Day5 with a large jump in gene expression at Day 7 analysis timepoint andstayed at this expression level for the rest of the study period.Expression of the neural stem cell marker Nestin also started to slowlyincrease from Day 5 onwards. Expression of the neuronal genesβIII-tubulin (TUBB3) and Map2b were slightly elevated already from Day 2onwards, but significantly increased on Day 5 onwards. Expression of amarker for acetylcholine receptors (found in neurons), acetylcholineesterase (ACHE), was also slightly elevated from Day 2 onwards, but didnot significantly increased until Day 7 onwards. It should be noted thatamong the neural stem cell markers that were analyzed, the relativeexpression of Sox2 was highly and early expressed which could then bedirectly or indirectly interact with the exogenous Msi/Ngn2 and/or othergenes in the activation of Nestin, GFAP, and endogenous Msi1 and Ngn2and other genes that promote the reprogramming and cell fate change, aswell as the activation of neuronal genes like βIII-tubulin (TUBB3),Map2b, and ACHE.

TABLE 9 RT-PCR analysis of exogenous and endogenous relative expressionof Msi1, Ngn2 and MBD2 from Day 2-10 after transfection of fibroblastcells with pCMV-Msi1-Ngn2(Msi1/Ngn2) and MBD2 and cultured for 10 daysin proliferation medium. Cells were collected at different time point toanalyse endogenous gene expression. Endogenous Endogenous EndogenousMSI1 MSI1 NGN2 NGN2 MBD2 MBD2 Rel. Std. Rel. Std. Rel. Std. Rel. Std.Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.Exp. Dev. #1 Day 12 Untransfected HFF 1.01 0.18 1.04 0.38 1.01 0.15 1.010.15 1.01 0.21 1.00 0.14 #2 Day 12 HFF Msi1/Ngn2 + 1102.17 91.80 620.5619.49 2208.36 375.09 51.09 14.69 1.09 0.00 0.83 0.06 MBD2 #3 Day 18 HFFMsi1/Ngn2 + 1470.36 164.35 950.07 152.50 71.57 52.59 122.66 39.63 1.210.02 0.73 0.08 MBD2 #4 Untransfected Keratinocytes 1.49 N/A 1.01 N/A1.00 N/A 1.00 N/A 1.02 N/A 1.00 N/A #5 Day 12 Keratinocytes 4142.78872.87 364.20 60.90 4656.42 232.63 102.01 3.18 0.40 0.14 0.74 0.30Msi1/Ngn2 + MBD2 #6 Day 18 Keratinocytes 4830.20 291.17 486.38 19.5950.01 6.99 43.08 13.78 0.40 0.01 0.67 0.01 Msi1/Ngn2 + MBD2 #7Untransfected CD34+ 1.01 0.19 1.00 0.01 1.01 0.16 1.17 0.87 1.00 0.021.00 0.07 #8 Day 18 CD34+ Msi1/Ngn2 + 3969.52 286.36 147.99 7.08 2.030.55 3.72 1.23 0.43 0.06 0.90 0.18 MBD2 hNPC (14-Oct-09, EXP0067)7574.57 234.74 1141.14 49.15 8.18 5.64 6.27 5.19 0.58 0.00 2.35 0.03

TABLE 10 RT-PCR analysis of relative expression of Nestin, Map2b, TUBB3,ACHE, GFAP, and Sox2 from Day 2-10 after transfection of fibroblastcells with pCMV-Msi1-Ngn2 (Msi1/Ngn2) and MBD2 and cultured for 10 daysin proliferation medium. Cells were collected at different time point toanalyse endogenous gene expression. NES MAP2 TUBB3 ACHE GFAP SOX2 Rel.Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Std. Exp. Dev. Exp. Dev.Exp. Dev. Exp. Dev. Exp. Dev. Rel. Exp. Dev. #1 Untransfected Day 2 1.000.04 1.00 0.01 1.00 0.03 1.00 0.08 1.01 0.23 1.17 0.87 #2 Msi1/Ngn2 +MBD2/+Noggin 0.88 0.01 8.59 0.18 1.38 0.03 5.71 1.06 4.56 0.08 1.26 0.82Day 2 #3 Untransfected Day 3 1.38 0.07 0.66 0.03 0.40 0.02 1.36 0.061.95 0.38 2.34 2.29 #4 Msi1-Ngn2 + MBD2/+Noggin 1.39 0.08 4.31 0.24 0.790.09 6.03 0.60 4.66 0.02 0.96 0.10 Day 3 #5 Untransfected Day 4 2.430.23 1.78 0.11 0.44 0.01 2.70 0.02 3.76 0.86 0.93 0.01 #6 Msi1/Ngn2 +MBD2/+Noggin 1.91 0.06 2.81 0.20 0.64 0.02 6.76 0.64 8.67 1.06 5.37 6.06Day 4 #7 Untransfected Day 5 1.40 0.05 1.13 0.04 0.41 0.03 1.17 0.375.44 0.02 15.03 8.77 #8 Msi1-Ngn2 + MBD2/+Noggin 4.31 0.08 71.60 6.431.34 0.01 7.60 0.18 42.28 2.94 66377.25 4089.77 Day 5 #9 UntransfectedDay 7 2.24 0.00 4.02 0.15 1.22 0.05 1.10 0.48 7.61 1.24 1.34 0.02 #10Msi1/Ngn2 + MBD2/+Noggin 3.07 0.11 48.10 2.85 2.70 0.05 13.11 1.303271.10 149.81 44255.59 2004.08 Day 7 #11 Untransfected Day 9 4.37 0.2314.55 0.96 1.75 0.14 3.35 0.36 15.95 0.23 429.09 119.98 #12 Msi1-Ngn2 +MBD2/+Noggin 7.97 0.16 123.55 3.27 2.79 0.12 16.59 0.03 3152.25 3.31114149.70 3372.20 Day 9 #13 Untransfected Day 10 3.48 0.44 10.03 0.371.63 0.21 3.20 0.81 5.64 1.92 14.66 5.03 #14 Msi1/Ngn2 + MBD2/+Noggin7.48 0.22 100.25 6.66 2.87 0.03 17.49 1.35 3374.03 22.47 101105.493996.44 Day 10

Example IV Comparison of the Nucleofector™® II Device and theNucleofector™® 96-Well Shuttle® Device in the Reprogramming of HFF intoNSLC in Adherent and Floating Conditions

HFF cells were cultured as described in Example I, and transfected usingthe Nucleofector™® II Device (Lonza) as previously described in ExampleII or the Nucleofector™® 96-well Shuttle® Device (Lonza). The HFFs wereharvested with TrypLE™ (Gibco), and 1×10⁶ cells/transfection with theNucleofector™® II Device for 10 min at 90 g and 6×10⁶ cells/transfectionwith the Nucleofector™® 96-well Shuttle® Device for 5 min at 80×g. Aftercentrifugation, the cell pellet was gently resuspended in either 1000 ofBasic Nucleofector™ Solution for the Nucleofector™® II or 200 of SESolution (Cell line kit SE, Lonza) for the Nucleofector™® 96-wellShuttle®. For the Nucleofector™® II Device, each 100 μl of cellsuspension was combined with 2 different mixes of plasmid DNA (sample 1was mixed with 2 μg of pCMV6-XL5-Msi1 and 2 μg pCMV6-XL5-MBD2, andsample 2 with 2 μg of Msi1/Ngn2 and 2 μg pCMV6-XL5-MBD2). Each cellsuspension was transferred into an Amaxa certified cuvette andtransfected with the appropriate program (U-023). Right aftertransfection, 900 μl of warm CDM1 medium was added to each cuvette andthe sample was transferred into a culture plate coated with Laminin(Stemgent, 10 μg/ml) at a cell density of 1×10⁵ to 1.5×10⁵ cells per cm²or into non-cell culture treated Petri dishes for neurosphere formation.The cells were incubated at 37° C., 5% CO₂ overnight. However for theNucleofector™® 96-well Shuttle® Device, the steps described before weresimilar with the following exceptions: the cell suspension was mixedwith 0.6 μg of each DNA of the same 2 DNA mixes, the cell suspension wastransferred to a well of a 96-well Nucleoplate™ (Lonza) and transfectedwith the program FF-130™. After transfection, 80 μl of warm CDM1 mediumwas added to each well and the samples were left for 10 min in theincubator prior to being transferred into a laminin coated plate ornon-cell culture treated Petri dishes at the same cell density aspreviously mentioned. For both devices, these steps were repeated foreach sample that was transfected. Prior to transfection cells werecultured in CDM1 as described in Example I. After 24 hours, the mediumwas switched to a mix of 75% CDM medium and 25% Proliferation Mediumwhich was supplemented with EGF (20 ng/ml), FGF-2 (20 ng/ml), Noggin (20ng/ml) and Cytochalasin B (10 μg/ml) and the cells were incubated at 37°C., 5% CO₂ and 5% O₂. The medium was changed daily with an increasedproportion of Neural proliferation medium up to 100% by Day 4 and adecreased proportion of Cytochalasin B that was completely omitted byDay 5. Forskolin (10 μM) was added to the medium from Day 4 onwards. Thecells in floating conditions were pelleted by centrifugation and theirmedium changed daily as described for the adherent condition. Cells werecollected at Day 3, 7, and 12 for immunohistochemistry analysis.

Fluorescence images were taken with a Cellomics™ ArrayScan HCS Reader™microscopy system to determine an estimate of the percentage of cellspositive for Sox2, a neural stem cell marker. This analysis revealedthat in untransfected controls and at 3 days after transfection, nonuclear Sox2 staining was detectable. However, at Day 7 and Day 12 thepercentage of Sox2 positive cells increased progressively under alltransfection conditions except the pCMV6-XL5-Musashi and pCMV6-XL5-MBD2Nucleofector™® II condition. The highest percentage at Day 12 wasobtained with Msi1/Ngn2 and pCMV6-XL5-MBD2 transfected with theNucleofector™® 96-well Shuttle® Device (˜80%). The same combinationtransfected with the Nucleofector™® II yielded only ˜35% positive cells.The pCMV6-XL5-Musashi and pCMV6-XL5-MBD2 with the Shuttle® produced ˜20%positive cells, while generally none were observed with theNucleofector™® II. The percentage of positive cells varied stronglybetween wells. The staining indicated that the cell population was nothomogenous, since fields of densely arranged Sox2 positive cells andcomplete fields with only negative cells could be found in all cases. Ingeneral the Shuttle® was initially more toxic to cells than theNucleofector™® II, however at least in the case of Msi1/Ngn2 andpCMV6-XL5-MBD2 shuttle, the Sox2 positive population rapidly expandedfrom Day 7 to Day 12 to have twice as many Sox2 positive cells ascompared to the Nucleofector™® II. The cells in floating conditions didnot form spheres during the 12 day experiment in any of the conditions,suggesting that the formation of neurospheres requires either thegeneration of neural stem-like cells in adherent conditions first ormore time.

Table 11 shows the percentage of Sox2 positive cells with a typicalneural stem cell morphology using both the Nucleofector™® II Device andthe Nucleofector™® 96-well Shuttle® Device. The latter had theadvantages of requiring a smaller starting material (less cells and lessDNA required) and in addition gave rise to a higher number of Sox2positive cells. Moreover a very small population of Sox2 positive cellswas observed with the Shuttle® Device only upon transfection with onlyone neurogenic transcription factor (Msi) in the presence of the DNAdemethylator MBD2.

TABLE 11 Percentage of positive cells for Sox2 after transfection offibroblast cells with different expression vectors. After transfectionthe cells were cultured in proliferation medium (StemCell Technologies)supplemented by EGF (20 ng/ml, Peprotech) and FGF (20 ng/ml, Peprotech)for two weeks at 37° C./5% CO₂/5% O₂. The percentage of immunopositivecells was determined by Cellomics ™ and represented as mean ± SD (n =3-5). % Sox2 positive cells Day 3 Day 7 Day 12 Total Total Total CellCell Cell Sox2 count Sox2 count Sox2 count Shuttle MSI1/NGN2 + MBD2 1.34± 6430 ± 31 ± 20 ± 10683 ± 78.17 ± 29341 ± 0.10 566 8.03 1112 3.10 2527Msi + MBD2 1.08 ± 8253 ± 3.19 ± 8953 ± 19.05 ± 11082 ± 0.61 399 3.57 67217.88 2999 Nucleofector ™ MSI1/NGN2 + MBD2 0.87 ± 21870 ± 14.30 ± 37321± 35.93 ± 33009 ± 0.30 4476 1.83 6877 7.10 1567 Msi + MBD2 0.64 ± 46793± 0.35 ± 34854 ± 0.51 ± 32095 ± 0.07 8808 0.16 2186 0.25 3236

Example V Neurosphere Formation Assay and Cell Differentiation Analysis

Based on previous studies showing that greater proportionalreprogramming is achieved by transfecting two neurogenic genes, thisstudy was designed to evaluate the number of reprogramming cells byusing the vector Msi1/Ngn2, containing two neurogenic transcriptionfactors (Msi1 and Ngn2) and the role of DNA demethylator or DNAmethylation inhibitor (5-azacytidine) and histone deacetylationinhibitor (VPA) in the reprogramming process.

HFFs were cultured and treated with cytochalasin B as described inExample III, and treated simultaneously with VPA (1 mM) and5-Azacytidine (0.5 μM). After two days of treatment, cells weretransfected by Nucleofection as described in Example II with theconstructed vector Msi1/Ngn2. After preparing the cells, they were mixedwith 2 μg of total DNA (Msi1/Ngn2) and cells that had not been treatedwith chemical inhibitors (VPA and 5-Aza) were co-transfect with MBD2 (2μg), using the appropriate program (U023). The samples were transferredinto a coated culture plate with Laminin (10 μg/ml, Sigma) and incubatedin a humidified 37° C./5% O₂/5% CO₂ incubator. The medium was changed tothe proliferation basal media, Neural Proliferation Medium (NeuroCult™proliferation Kit, StemCell Technologies), with the presence of Noggin(20 ng/ml, Peprotech), recombinant hFGF (20 ng/ml, Peprotech), andrecombinant hEGF (20 ng/ml, Peprotech). Following 6 days oftransfection, cells were harvested using Accutase™ (Millipore),centrifuged (300×g, 5 min, RT) and plated in uncoated cell culturedishes in NeuroCult™ NSC Proliferation medium to investigate thecapacity to grow cells in suspension as neurospheres or on Laminincoated-plates for adherent culture. To prevent loss of floating spheresduring media changes, cells were sedimented by centrifugation at 150×gfor 3 min at room temperature (RT). The pellet was then resuspended infresh medium and plated into new uncoated, low-bind cell culture dishes.Cultures were incubated at 37° C., 5% CO₂, 5% O₂ and were fed daily forat least two months.

To investigate whether a single cell from human neural precursor cells(hNPCs) and human NSLCs was able to generate a neurosphere (a standardtest for proving that a cell is a neural stem cell), neurospheres weredissociated into single cells and these single cells were isolated andcultured in proliferation medium in suspension, and neurosphereformation was monitored by taking bright field images using lightmicroscope (Nikon, 10×) and by Cellomics™. These cells started toproliferate and grew as spheres starting day 6 to day 10 (FIG. 4A).Immunohistochemistry analysis of these spheres (Table 12 and FIG. 4) onDay 20, revealed immunopositive staining for the neural stem cellsmarkers Sox2, Musashi, CD133, Nestin, and GFAP. Cells also stainedpositive for βIII-tubulin (a marker for neurons), 04 (a marker foroligodendrocytes), and GFAP (a marker for astrocytes), indicating thetri-potent differentiation potential of both sets of cells (NSLC andhNPC), and negative for NGFrec and NeuN (markers for differentiatedneurons) indicating that the cells were not terminally differentiated.

TABLE 12 Percentage of positive cells for neural stem cells, andneuronal, astrocyte and oligodendrocyte lineage markers in neurospheresformed from single NSLCs and hNPCs cultured in proliferation medium(StemCell Technologies) supplemented by EGF (20 ng/ml, Peprotech) andFGF (20 ng/ml, Peprotech) for 20 days at 37° C./5% CO₂/5% O₂. Thepercentage of positive cells was determined by Cellomics ™ andrepresented as mean ± SD. % of positive cells NSLCs hNPCs Musashi 91.8 ±6.8 88.6 ± 7.9 Nestin 78.6 ± 5.7  75.4 ± 12.0 GFAP 69.2 ± 7.4 78.6 ± 8.4βIII-tubulin 85.6 ± 6.4 76.6 ± 8.4 P75 0 0 NeuN 0 0 O4 65.4 ± 6.6 71.4 ±7.5 CD133 0 0

HFF cells were cultured as described in Example I, and transfected usingthe Nucleofector™ II device (Lonza) as described in Example II. Cellswere co-transfected with pCMV6-XL5-Msi/pCMV6-XL4-Ngn2, pCMV-Msi1-Ngn2with MBD2 or pre-treated with VPA/5aza. Cells were cultured inproliferation medium as suspension or adherent cultures. Gene expressionanalysis on 8 samples was performed as previously described in Example Iwith the customized Neuronal Markers 2 TLDA (Table 13) which profiledthe expression of 48 genes (including three housekeeping genes: ACTIN,GAPDH and PPIA) in four major categories; 1) fibroblast specific genes;2) neuronal lineage specific genes; 3) Neural stem cell marker specificgenes; and 4) Genes for growth factors and their receptors.

TABLE 13 Neuronal Markers 2 TLDA Layout (Applied Biosystems) GeneSymbols 1 2 3 4 5 6 7 8 9 10 11 12 1 ACTB PPIA COL3A1 LOX SI00A4 SYT1SNAP25 NEUROD1 MBP NKX2-2 GAPDH OLIG2 2 VIM SOX3 SOX9 PROM1 SOX1 SOX2KLF4 POU5F1 STAT3 PIK3CG GDNF NGF 3 ACTB PPIA COL3A1 LOX SI00A4 SYT1SNAP25 NEUROD1 MBP NKX2-2 GAPDH OLIG2 4 VIM SOX3 SOX9 PROM1 SOX1 SOX2KLF4 POU5F1 STAT3 PIK3CG GDNF NGF 5 ACTB PPIA COL3A1 LOX SI00A4 SYT1SNAP25 NEUROD1 MBP NKX2-2 GAPDH OLIG2 6 VIM SOX3 SOX9 PROM1 SOX1 SOX2KLF4 POU5F1 STAT3 PIK3CG GDNF NGF 7 ACTB PPIA COL3A1 LOX SI00A4 SYT1SNAP25 NEUROD1 MBP NKX2-2 GAPDH OLIG2 8 VIM SOX3 SOX9 PROM1 SOX1 SOX2KLF4 POU5F1 STAT3 PIK3CG GDNF NGF 9 ACTB PPIA COL3A1 LOX SI00A4 SYT1SNAP25 NEUROD1 MBP NKX2-2 GAPDH OLIG2 10 VIM SOX3 SOX9 PROM1 SOX1 SOX2KLF4 POU5F1 STAT3 PIK3CG GDNF NGF 11 ACTB PPIA COL3A1 LOX SI00A4 SYT1SNAP25 NEUROD1 MBP NKX2-2 GAPDH OLIG2 12 VIM SOX3 SOX9 PROM1 SOX1 SOX2KLF4 POU5F1 STAT3 PIK3CG GDNF NGF 13 ACTB PPIA COL3A1 LOX SI00A4 SYT1SNAP25 NEUROD1 MBP NKX2-2 GAPDH OLIG2 14 VIM SOX3 SOX9 PROM1 SOX1 SOX2KLF4 POU5F1 STAT3 PIK3CG GDNF NGF 15 ACTB PPIA COL3A1 LOX SI00A4 SYT1SNAP25 NEUROD1 MBP NKX2-2 GAPDH OLIG2 16 VIM SOX3 SOX9 PROM1 SOX1 SOX2KLF4 POU5F1 STAT3 PIK3CG GDNF NGF 13 14 15 16 17 18 19 20 21 22 23 24 1ALDHIL1 DIO2 GFAP NCAM1 FOXJ1 PDGFRA MKI67 NES CSPG4 DLX2 MSI1 CROCC 2BDNF CNTFZFP91- GAP43 NRG1 NPY CSF3 BMP4 TGFB1 VEGFA NGFR EGFR KDR CNTF3 ALDHIL1 DIO2 GFAP NCAM1 FOXJ1 PDGFRA MKI67 NES CSPG4 DLX2 MSI1 CROCC 4BDNF CNTFZFP91- GAP43 NRG1 NPY CSF3 BMP4 TGFB1 VEGFA NGFR EGFR KDR CNTF5 ALDHIL1 DIO2 GFAP NCAM1 FOXJ1 PDGFRA MKI67 NES CSPG4 DLX2 MSI1 CROCC 6BDNF CNTFZFP91- GAP43 NRG1 NPY CSF3 BMP4 TGFB1 VEGFA NGFR EGFR KDR CNTF7 ALDHIL1 DIO2 GFAP NCAM1 FOXJ1 PDGFRA MKI67 NES CSPG4 DLX2 MSI1 CROCC 8BDNF CNTFZFP91- GAP43 NRG1 NPY CSF3 BMP4 TGFB1 VEGFA NGFR EGFR KDR CNTF9 ALDHIL1 DIO2 GFAP NCAM1 FOXJ1 PDGFRA MKI67 NES CSPG4 DLX2 MSI1 CROCC10 BDNF CNTFZFP91- GAP43 NRG1 NPY CSF3 BMP4 TGFB1 VEGFA NGFR EGFR KDRCNTF 11 ALDHIL1 DIO2 GFAP NCAM1 FOXJ1 PDGFRA MKI67 NES CSPG4 DLX2 MSI1CROCC 12 BDNF CNTFZFP91- GAP43 NRG1 NPY CSF3 BMP4 TGFB1 VEGFA NGFR EGFRKDR CNTF 13 ALDHIL1 DIO2 GFAP NCAM1 FOXJ1 PDGFRA MKI67 NES CSPG4 DLX2MSI1 CROCC 14 BDNF CNTFZFP91- GAP43 NRG1 NPY CSF3 BMP4 TGFB1 VEGFA NGFREGFR KDR CNTF 15 ALDHIL1 DIO2 GFAP NCAM1 FOXJ1 PDGFRA MKI67 NES CSPG4DLX2 MSI1 CROCC 16 BDNF CNTFZFP91- GAP43 NRG1 NPY CSF3 BMP4 TGFB1 VEGFANGFR EGFR KDR CNTF Sample information Sample ID Sample Name TLDA Port 1HFF Ctrl 1 2 ReNcell Undifferentiated Ctrl 2 3 Msi1-Ngn2/MBD2 3 4Msi1-Ngn2/MBD2 4 5 Msi1-Ngn2/VPA + AZA 5 6 Msi1-Ngn2 6 7 Msi1-Ngn2/MBD2,neurospheres 7 8 Msi1-Ngn2/MBD2, neurospheres 8

As shown in Table 14, fibroblast-specific genes (Col3A1, Lox, S100A4)were down-regulated in reprogrammed cells, indicating the loss offibroblast-specific genes following transfection (note that not allcells got transfected and reprogrammed, so the presence offibroblast-specific gene expression in the cultures is mostly from theun-programmed fibroblasts left in the culture). The expression of thesegenes is observed to increase when HFFs were transfected in the absenceof DNA demethylator or the DNA methylation inhibitor, indicating thatdown-regulation of differentiated markers of fibroblast cells requiresDNA demethylation. The expression of ectoderm genes such as Msi1, Sox2,and Nestin was remarkably increased following transfection inconjunction with DNA demethylation. The expression of neuronal markers,such as synaptogamin1 (a synaptic vesicle protein) and NeuroD1 wasup-regulated in transfected cells with Msi1/Ngn2/MBD2, and slightlyincreased in transfected cells with Msi1/Ngn2/VPA and 5-AZA. Theselected three markers of oligodendrocytes were detected in thetransfected cells with a strong increase of Olig2. Two markers forastrocytes, GFAP and ALDH1L1, were enhanced following transfection. Theresults support the idea that neurospheres are composed of heterogeneousprogenitor subtypes.

Among the neurotrophic factors, expression of CNTF was slightlyincreased in the reprogrammed cells. The expression of GAP-43 andneuropeptide Y (NPY) were the most annotated genes. GAP-43 has long beenacknowledged to play a pivotal role in axonal plasticity and is used asa marker of regenerating neurite outgrowth and synaptogenesis, both inembryonic development and in neuronal regeneration in injured brain andspinal cord. Expression of receptors for growth and neurotrophic factorswas increased, such as neurotrophic receptor tyrosine kinase expression.

TABLE 14 Gene array analysis was performed after one month oftransfection of human fibroblast cells with Msi1/Ngn2, in the presenceMBD2 or VPA and 5-Aza. Cells were cultured on coated culture plates asadherent cells or on untreated culture plates as neurospheres inproliferation medium (StemCell Technologies) supplemented with EGF (20ng/ml) and FGF (20 ng/ml). Untransfected cells were considered asnegative control and ReNcell (Millipore) as positive control. Relativeexpression to #1 HFF Ctrl #5 #8 #3 #4 Msi1- #7 Msi1-Ngn2/ #2 Msi1- Msi1-Ngn2/ #6 Msi1-Ngn2/ VPA + ReNcell Ngn2/ Ngn2/ VPA + Msi1- MBD2, AZA,Symbol Common name and description Undiff MBD2 MBD2 AZA Ngn2neurospheres neurospheres Fibroblast/ECM component COL3A1 Collagen, typeIII, alpha 1, 0.00 0.03 0.02 0.02 11.92 0.00 0.00 fibroblast marker LOXLysyl oxidase, ECM component 0.01 0.03 0.01 0.01 2.38 0.00 0.00 FSP1Fibroblast transcription site-1, 0.04 0.04 0.06 0.05 3.22 0.05 0.05enzyme for ECM remodeling Neuron markers SYT1 Synaptotagmin1, a synapticvesicle 106.49 108.40 78.66 26.72 22.42 37.61 16.80 protein in neuronsSNAP25 SNAP25, mature neuron marker 4.72 6.10 7.89 3.11 3.19 6.47 4.00NEUROD1* Neurogenic differentiation 1, 2.32 93.35 100.84 2.02 3.11271.11 10.23 neuron marker Oligodendrocyte markers MBP* Myelin BasicProtein, mature 2.32 48.53 18.11 6.94 667.56 16.67 1.67 oligodendrocytemarker NKX2-2* NK2 homeobox 2, remyelination 2.32 75.31 54.65 1.66 3.111.67 1.74 OLIG2* Oligodendrocyte lineage 2856.4 15594 67369 38733 3.1192420 101733 transcription factor 2, oligodendrocyte progenitorAstrocyte markers ALDH1L1* Aldehyde dehydrogenase 1 family 6.20 3.774.65 1.66 0.02 5.87 9.59 member L1, astrocyte DIO2* Deiodinaseiodothyronine type II, 23.20 0.00 0.00 0.00 0.51 0.00 0.00 astrocytemarker GFAP Glial fibrillary acidic protein, 3342.1 6899.0 6291.0 4800.91.27 3118.7 3222.0 astrocyte marker NSCS markers NCAM1 NCAM1, neuroblastmarker 23.21 43.90 24.45 12.72 1.13 31.93 36.70 PDGFRA Plate-derivedgrowth factor 0.05 0.01 0.01 0.00 4.42 0.00 0.01 receptor alpha,oligodendrocyte progenitor cells NES Nestin, neural progenitor 5.7619.84 19.56 3.46 4.23 16.57 8.36 MSI1*, ** Musashi I, neuroblast marker5120.3 5985.2 5262.7 5645.1 204.34 3179.6 4113.6 SOX1* Sox1, neuralprogenitor 679.21 223.59 373.14 361.67 3.11 287.82 323.23 SOX2* Sox2,NSCs 1924084 2265299 1889166 1014816 3.11 1313765 1103212 Neurotrophic/Growth Factor GDNF* Glial cell derived neurotrophic 0.01 0.02 0.02 0.001.69 0.00 0.00 factor NGF* Nerve growth factor 0.00 0.00 0.00 0.00 1.480.00 0.00 BDNF Brain derived neurotrophic factor 0.03 0.09 0.09 0.050.82 0.02 0.01 CNTF* Ciliary neurotrophic factor 9.25 4.32 3.11 2.9064.05 2.31 3.39 GAP43 Growth associated protein 43, 917.52 3506.5 1530.8452.75 584.00 746.25 578.52 neural regeneration NRG1* Neuregulin 1,neural regeneration 0.01 0.00 0.00 0.00 0.40 0.00 0.00 NPY* NeuropeptideY, interneuron 2.32 675.69 465.04 153.54 3.11 1244.0 130.38 CSF3* Colonystimulating factor 3, neural 0.50 0.03 0.02 0.58 18.62 0.02 0.02regeneration BMP4 Bone morphogenetic protein 4, 0.83 0.26 0.74 0.4511.03 0.09 0.07 remyelination marker TGFB1 Transforming growth factor,beta 1 0.85 2.39 0.92 0.83 0.65 0.45 0.58 Angiogenesis VEGFA Vascularendothelial growth factor 2.77 14.93 15.01 2.67 3.82 2.80 3.21Neurotrophin/ Growth Factor Receptors NGFR/P75 NGFR, neurotrophinreceptor 5.35 3.29 5.78 9.10 7.53 7.26 17.51 EGFR Epidermal growthfactor receptor 0.89 0.77 0.86 0.79 1.63 1.44 1.25 KDR* Kinase insertdomain receptor, 210.87 259.42 263.45 51.85 0.07 11.23 17.50 growthfactor receptor

Further analysis and quantification of the adherent population of NSLCsshowed that cells were positively stained for Sox2 (93.43±1.9%), nestin(60.76±5.7%), and GABA (37.48±4.9), while these markers wereundetectable in untransfected cells (FIG. 5, Table 15). Furthermore,these cells stained positive for p75NTR (31.15±1.6), βIII-tubulin(37.55±0.6%) and GFAP (16.47±0.9). However, untransfected HFFs onlystained positive for HFF markers (FIG. 5), such as fibronectin andfibroblast protein marker, while these markers were undetectable inreprogrammed cells, demonstrating that the reprogrammed cells lostmarkers of the original cells and adopted morphology and markers ofneural stem cells and a neuronal lineage.

TABLE 15 The percentage of cells stained positive for neural stem cellmarkers and fibroblast markers in untransfected cells and transfectedcells with pMsi1/Ngn2/MBD2. Transfected cells (NSLCs) possess a highpercentage of neural stem markers but a very low percentage offibroblast markers as compared to untransfected cells. The percentage ofimmunopositive cells was determined by Cellomics ™ and represented asmean ± SD (n = 5). Transfected Untransfected fibroblast cells fibroblastcells (% of average positive (% of average positive Marker protein cells± stdv) cells ± stdv) Sox2 93.43 ± 1.9 1.90 ± 0.5 Nestin 60.76 ± 5.70.84 ± 0.2 p75NTR 31.15 ± 1.6 3.95 ± 1.7 NCAM 26.84 ± 3.8 0.87 ± 0.2S100 41.80 ± 0.6 1.60 ± 0.3 GFAP 16.47 ± 0.9 3.84 ± 0.9 βIII-Tubulin37.55 ± 0.6 1.90 ± 0.9 GABA 37.48 ± 4.9 2.54 ± 0.5 Fibronectin  1.05 ±0.7 94.19 ± 0.9  Fibroblast marker protein  4.81 ± 1.0 50.30 ± 7.8 

This study showed as well that NSLCs have the capacity to proliferate inculture and exhibit stable morphology, gene and protein expression thatwere maintained for the entire study period, which was for over fivemonth in culture (Table 16).

TABLE 16 Doubling time of NSLCs over serial passages. NSLCs weremaintained in proliferation conditions for 35 passages in a 37° C., 5%CO₂ and 5% O₂ incubator. The time required for the cell population todouble (g) was calculated for each passage, and was defined as g =(ln2)/k, where k was the number of generations that occured per unittime (t) defined as, k = (ln N_(f) − ln N₀)/t, where N_(f) was the finalcell number and N₀ the initial seeded cell number. The averagegeneration time was 25.4 h over 35 passages. Passage Time LN LN k gnumber (h) N₀ N_(f) (h⁻¹) (h) 2 168 11.513 15.577 0.024 38.655 3 21611.513 16.195 0.022 31.977 4 192 11.513 18.258 0.035 39.730 5 144 11.51316.258 0.033 21.036 6 144 11.513 16.258 0.033 21.036 7 144 11.513 15.7020.029 33.824 8 168 11.513 15.870 0.026 26.729 9 120 11.513 16.811 0.03132.548 10 144 11.513 15.415 0.027 35.580 11 120 13.122 15.895 0.023 3012 120 11.513 15.747 0.035 19.645 13 168 11.513 15.870 0.026 26.729 14168 12.429 15.870 0.020 23.847 15 168 11.513 15.520 0.024 29.059 16 19211.513 16.167 0.024 28.596 17 144 11.513 15.239 0.026 36.791 18 16811.513 15.790 0.025 37.229 19 120 13.122 15.870 0.023 30.276 20 14413.122 16.249 0.022 31.922 21 96 13.122 15.761 0.027 25.214 22 12013.122 15.870 0.023 30.276 23 120 13.122 15.761 0.022 31.518 24 9613.122 15.687 0.027 25.943 25 96 13.122 16.013 0.030 23.022 26 96 13.12216.067 0.031 22.599 27 96 13.122 16.300 0.033 20.938 28 120 13.12216.482 0.028 24.752 29 96 13.122 16.380 0.034 20.424 30 96 13.122 16.3000.033 19.938 31 120 13.122 16.483 0.028 22.752 32 96 13.122 16.062 0.03120.640 33 96 13.122 16.300 0.033 20.938 34 96 13.122 16.077 0.031 15.51935 96 13.122 16.077 0.031 15.519

Gene Expression Microarray

Microarray expression analysis was performed to get a global overview tocompare the gene expression profile of passage 7 NSLC to both HFF (thecells that the NSLC were created from) and hNPCs. NSLC (n=3), HFF (n=2),and hNPC (n=3) were resuspended in RNAlater™ (Qiagen) and shipped toGenotypics (India) where the samples were processed and the GeneExpression Microarray was performed.

In brief, Genotypics extracted RNA from the samples and performedQuality Control using an Agilent Bioanalyzer™. Labelling was done usingAgilent's Quick Amp™ kit (cDNA synthesis and in vitro transcription),followed by Labelling QC. Hybridization was then performed using the8×60K array, and scanning was done using high throughput Agilent scannerwith SureScan™ technology. The Agilent Feature Extraction software wasused for automated feature extraction, followed by Raw Data QC and ImageQC. Advanced Data Analysis was then performed, including Pathway andGene Ontology analysis using Agilent's GeneSpring GX™ v10.0 andGenotypic's Biointerpreter Software. The NSLC samples were compared tothe HFF samples (Set 1) and hNPC samples (Set 2) The NSLC samples had aglobal gene expression pattern that was much closer to the hNPCs thanthe HFFs from which the NSLCs were created (FIG. 23). Pearsoncorrelation analysis revealed that NSLCs are closely related to hNPCs,including in terms of neuronal lineage markers, regenerative genes andmigration genes. These data confirm that NSLCs are similar, but notidentical, to hNPCs.

Microarray analysis revealed an up-regulation of neural precursor genesin the NSLC samples as compared to the HFF samples. ACTL6A and PHF10,which both belong to the neural progenitors-specific chromatinremodelling complex (npbaf complex) and are required for theproliferation of neural progenitors, were up-regulated by 2.9-fold and2.3 fold respectively. MSI2, which plays a role in the proliferation andmaintenance of stem cells in the central nervous system, wasup-regulated by 6-fold (Table X1). Glia genes were up-regulated in theNSLC samples as compared to the HFF samples. GFAP, is a neural stemcell- and astrocyte-specific marker that, during the development of thecentral nervous system, distinguishes astrocytes from other glial cells,is highly up-regulated in the NSLC sample as compared to HFF (690-fold).OLIG1, which promotes formation and maturation of oligodendrocytes,especially within the brain, is also highly up-regulated in NSLC sampleas compared to HFF (370-fold) (Table X2).

Table X3 lists a subset of regenerative genes that are up-regulated inthe NSLC samples as compared to the HFF samples. SOX2, a gene criticalfor early embryogenesis and for embryonic stem cell pluripotency as wellas neural stem cells, is highly up-regulated in the NSLC samples ascompared to the HFF samples (5000-fold). CCND2, which is essential forthe control of the cell cycle at the G1/S (start) transition, is alsoup-regulated in NSLC samples (70-fold as compared to HFF samples). Asshown in Table X4, numerous fibroblast genes were down-regulated in theNSLC samples as compared to the HFF samples. This shows that the NSLClose the expression of numerous fibroblast genes as it gets reprogrammedfrom HFF to NSLC.

Table X5 show that neural precursor genes were also up-regulated in theNSLC samples as compared to the hNPC samples. BDNF, which promotes thesurvival and differentiation of selected neuronal populations of theperipheral and central nervous systems during development, is even morehighly expressed in NSLC samples than in hNPC samples (34-foldup-regulation). Table X6 shows that a subset of Glia genes are alsoup-regulated in the NSLC samples as compared to the hNPC samples. GFAP,a neural stem cell- and astrocyte-specific marker that, during thedevelopment of the central nervous system, distinguishes astrocytes fromother glial cells, is more highly expressed in NSLC samples than hNPCsamples (13-fold). PLP1, the major myelin protein of the central nervoussystem which plays an important role in the formation or maintenance ofthe multilamellar structure of myelin, is also more highly expressed inNSLC samples than in hNPC samples (20-fold).

Regenerative genes were also up-regulated in the NSLC samples ascompared to the hNPC samples (Table X7). BMP2, a neural crest marker,but which induces growth especially of cartilage and bone formation andBMP4, which in turn induces cartilage and bone formation and acts inmesoderm induction, tooth development, limb formation and fracturerepair, but also in neural stem cells, were both more highly expressedin NSLC samples than in hNPC samples (18-fold and 20-fold respectively).GAP43, which is a major component of the motile growth cones that formthe tips of elongating axons was more highly expressed in NSLC samplesthan hNPC samples (4-fold). This suggests the regenerative potential ofNSLC. HOXB4, a transcription factor that is involved in development andalso in the expansion of neural stem cells as well as hematopoietic stemand progenitor cells in vivo and in vitro making it a potentialcandidate for therapeutic stem cell expansion, was also more highlyexpressed in NSLCs than in hNPCs. This data indicates that NSLCs aremore ‘stem-like’ or have more ‘stemness’ than hNPCs.

TABLE X1 Up-regulated Neural Precursor genes (NSLC vs. HFF) AccessionFold change of NSLC GeneSymbol Number compared to HFF¹ p-value ACTL6ANM_178042 2.90 0.000 ADAM9 NM_001005845 2.64 0.004 AIFM1 NM_004208 2.450.000 BCAT1 NM_005504 3.23 0.000 BMP2 NM_001200 17.49 0.000 DLL1NM_005618 40.32 0.000 EDNRB NM_003991 933.03 0.000 ERBB4 NM_005235 53.220.006 GMNN NM_015895 4.42 0.000 HES5 BC087840 102.33 0.000 KIF1BNM_015074 9.45 0.002 LIMK1 NM_002314 2.44 0.002 MAPK8IP1 NM_005456 5.880.001 MCHR1 NM_005297 68.19 0.001 MEF2C NM_002397 2.91 0.000 MSI2NM_170721 6.76 0.000 NMB NM_021077 3.65 0.000 NOS2A NM_000625 279.450.000 NOTCH1 NM_017617 6.75 0.000 NPAS3 NM_022123 187.85 0.000 PHF10NM_018288 2.28 0.001 PHLPP NM_194449 8.84 0.000 SMAD1 NM_005900 4.740.000 SNTG1 AL161971 34.05 0.000 SP8 NM_198956 1392.67 0.000 STAU2AK002152 3.35 0.000 STIL NM_003035 4.94 0.003 ¹Fold change representsthe up-regulation of the gene in the NSLC samples as compared to the HFFsamples. (n = 2 for HFF samples, n = 3 for NSLC samples).

TABLE X2 Up-regulated Glia genes (NSLC vs. HFF) Accession Fold change ofNSLC GeneSymbol Number compared to HFF¹ p-value ASTN1 NM_004319 51.440.000 ATP1B2 NM_001678 186.64 0.000 B3GAT1 NM_018644 1784.49 0.000 BCL2NM_000633 2.65 0.002 BMP7 NM_001719 41.35 0.000 CA14 NM_012113 43.440.000 CLCN2 NM_004366 4.18 0.000 CNDP1 NM_032649 4.39 0.010 CP NM_00009693.08 0.002 CXCR4 NM_001008540 4124.29 0.000 ERBB4 NM_005235 53.22 0.006FABP7 NM_001446 18702.36 0.000 GAB1 NM_207123 2.44 0.001 GFAP NM_002055696.51 0.000 GJB2 NM_004004 13.89 0.001 ITGB8 NM_002214 8.48 0.005KCNJ10 NM_002241 263.42 0.000 LMO3 NM_018640 194.32 0.000 MAP6D1NM_024871 3.99 0.000 MAPT NM_016835 2.38 0.001 NDE1 NM_017668 2.21 0.002NEFL NM_006158 10.30 0.001 NKX6-2 NM_177400 10.83 0.026 NOVA2 NM_0025167.51 0.000 NTN1 NM_004822 5.29 0.015 NTRK3 NM_001012338 15.32 0.000OLIG1 NM_138983 372.11 0.000 OLIG2 NM_005806 163.20 0.000 PARD6ANM_016948 4.12 0.001 PASK NM_015148 3.89 0.001 PAX6 NM_001604 28.530.001 PDCD11 ENST00000369797 2.23 0.001 PDE6B NM_000283 5.55 0.001 PER1NM_002616 2.43 0.001 PLP1 M54927 351.09 0.000 PTK2 NM_153831 4.22 0.000QKI NM_206855 8.75 0.003 S100B NM_006272 456.00 0.000 SLC1A3 NM_00417249.49 0.000 SORL1 NM_003105 27.61 0.000 SOX9 NM_000346 27.82 0.000 SPRY2NM_005842 15.83 0.000 TARDBP NM_007375 2.69 0.005 TSPAN12 NM_012338259.78 0.000

TABLE X3 Up-regulated Regenerative genes (NSLC vs. HFF) Accession Foldchange of NSLC GeneSymbol Number compared to HFF¹ p-value BMP2 NM_00120017.49 0.000 CCND2 NM_001759 72.79 0.000 DLL1 NM_005618 40.32 0.000 EGR1NM_001964 2.19 0.000 GAL NM_015973 25.93 0.000 GAP43 NM_002045 1297.420.000 HOXB4 NM_024015 102.34 0.000 NFE2L2 AF323119 2.80 0.004 NOTCH1NM_017617 6.75 0.000 PRPH NM_006262 6.44 0.000 SEMA3A NM_006080 3.030.004 SEMA6A NM_020796 23.58 0.000 SOX2 NM_003106 5165.92 0.000

TABLE X4 Down-regulated Fibroblast genes (NSLC vs. HFF) Accession Foldchange of NSLC GeneSymbol Number compared to HFF¹ p-value ACOT2NM_006821 0.30 0.000 AEBP1 NM_001129 0.16 0.001 AGA NM_000027 0.35 0.000ANXA2 NM_001002857 0.26 0.029 AP4E1 NM_007347 0.30 0.008 APOE NM_0000410.08 0.000 ARHGDIB NM_001175 0.24 0.009 ASAH1 NM_004315 0.31 0.000BDKRB1 NM_000710 0.00 0.001 BDKRB2 NM_000623 0.00 0.000 BDNF NM_1707350.12 0.000 BMP4 NM_001202 0.28 0.001 C3 NM_000064 0.25 0.001 C5orf13NM_004772 0.18 0.000 CACNA1C NM_000719 0.03 0.000 CASP4 NM_033306 0.000.000 CASP5 NM_004347 0.00 0.001 CCL2 NM_002982 0.20 0.000 CD36NM_001001547 0.07 0.023 CDC42EP2 NM_006779 0.06 0.000 CDC42EP3 NM_0064490.41 0.000 CDC42EP5 NM_145057 0.41 0.040 CDH11 NM_001797 0.00 0.000CEMP1 AL833099 0.30 0.001 CFH NM_001014975 0.01 0.010 CITED2 NM_0060790.14 0.000 COL12A1 NM_004370 0.00 0.001 COL1A1 NM_000088 0.01 0.000COL1A2 NM_000089 0.00 0.001 COL3A1 NM_000090 0.00 0.001 COL5A1 NM_0000930.00 0.000 CPT1A NM_001876 0.16 0.002 CROT NM_021151 0.27 0.002 CTSANM_000308 0.10 0.000 CTSB NM_147780 0.11 0.001 CXCL1 NM_001511 0.010.003 CXCL12 NM_000609 0.00 0.001 CYP27A1 NM_000784 0.28 0.011 CYR61NM_001554 0.10 0.000 DCHS1 NM_003737 0.29 0.000 DMPK NM_004409 0.360.000 DPT NM_001937 0.05 0.006 EFEMP1 NM_004105 0.00 0.000 ELN NM_0005010.13 0.001 EMX2 NM_004098 0.00 0.001 EPS8 NM_004447 0.18 0.000 ETS1NM_005238 0.15 0.003 FAH NM_000137 0.17 0.000 FAM14A NM_032036 0.220.001 FAP NM_004460 0.00 0.000 FBLN2 NM_001004019 0.18 0.000 FBN1NM_000138 0.01 0.002 FGF1 NM_000800 0.20 0.004 FGF13 NM_004114 0.040.006 FGF2 NM_002006 0.06 0.000 FGF5 NM_004464 0.01 0.003 FGF7 NM_0020090.04 0.001 FGF9 NM_002010 0.01 0.000 FGFR1 NM_023110 0.34 0.026 FHL2NM_201555 0.11 0.000 FN1 NM_212482 0.00 0.001 FSTL1 NM_007085 0.09 0.000GADD45B NM_015675 0.09 0.001 GALNT6 NM_007210 0.13 0.001 GAS6 NM_0008200.02 0.000 GBA NM_001005749 0.22 0.002 GBAP NR_002188 0.19 0.000 GCH1NM_000161 0.22 0.001 GGTA1 NR_003191 0.28 0.013 GIT2 NM_057169 0.370.003 GJA1 NM_000165 0.46 0.001 GLIS1 NM_147193 0.02 0.000 GM2A AK1279100.25 0.010 GNS NM_002076 0.29 0.000 GPC3 NM_004484 0.22 0.038 GREM1NM_013372 0.00 0.011 GSTM1 NM_146421 0.27 0.001 HAAO NM_012205 0.430.001 HERPUD1 NM_014685 0.19 0.000 HEXA NM_000520 0.24 0.000 HEXBNM_000521 0.36 0.000 HGF NM_001010932 0.09 0.028 HGS NM_004712 0.260.029 HIF1A NM_181054 0.36 0.005 HLA-A NM_002116 0.31 0.002 HLA-HNR_001434 0.19 0.001 HOXB13 NM_006361 0.03 0.004 HR NM_005144 0.18 0.002HSPG2 NM_005529 0.19 0.004 IDUA NM_000203 0.16 0.000 IGF1 NM_000618 0.100.004 IGFBP7 NM_001553 0.28 0.040 IKBKG NM_003639 0.42 0.001 IRF1NM_002198 0.28 0.002 ITGA1 NM_181501 0.00 0.001 ITGB3 NM_000212 0.050.000 KLF4 NM_004235 0.05 0.002 LEP NM_000230 0.07 0.001 LEPRE1NM_022356 0.24 0.000 LMNA NM_005572 0.42 0.000 LOX NM_002317 0.01 0.000LOXL4 NM_032211 0.10 0.003 LRRC8C NM_032270 0.15 0.013 MAGEL2 AJ2435310.31 0.002 MAN2B1 NM_000528 0.45 0.006 MAP3K8 NM_005204 0.27 0.001 MEIS2NM_170677 0.00 0.001 MKNK1 NM_003684 0.37 0.005 MMP1 NM_002421 0.000.000 MMP14 NM_004995 0.07 0.001 MMP2 NM_004530 0.04 0.000 MMP3NM_002422 0.00 0.001 MOXD1 NM_015529 0.24 0.000 MRAS NM_012219 0.150.001 MSX2 NM_002449 0.15 0.031 MTHFR NM_005957 0.27 0.014 MYC NM_0024670.05 0.000 MYL6 NM_079423 0.33 0.001 MYL9 NM_181526 0.01 0.000 NAGLUNM_000263 0.23 0.000 NBL1 NM_182744 0.11 0.000 NEK9 NM_033116 0.41 0.001NF2 NM_181831 0.46 0.000 NPC1 NM_000271 0.34 0.000 OPTN NM_0010082110.04 0.000 P4HB NM_000918 0.37 0.001 PALLD NM_016081 0.29 0.001 PAPPANM_002581 0.05 0.000 PCDHGB4 NM_032098 0.28 0.001 PCK2 NM_004563 0.040.000 PCOLCE NM_002593 0.00 0.000 PDGFRA NM_006206 0.02 0.010 PEX14BC017848 0.48 0.000 PFKL NM_001002021 0.35 0.004 PPARG NM_138711 0.010.000 PPFIBP2 NM_003621 0.08 0.000 PRR5 NM_015366 0.23 0.022 PSEN2NM_012486 0.34 0.002 PTGS1 NM_000962 0.29 0.000 PXDN AF200348 0.12 0.000PYCARD NM_013258 0.03 0.000 QSOX1 NM_002826 0.09 0.000 RASSF1 NM_1707130.30 0.001 RBMS1 NM_002897 0.14 0.001 RECK NM_021111 0.07 0.000 RETNM_020975 0.35 0.015 RFPL1S NR_002727 0.22 0.039 ROD1 NM_005156 0.370.001 RSU1 NM_012425 0.41 0.002 S100A4 NM_002961 0.03 0.000 SAMD9NM_017654 0.07 0.007 SCARB2 NM_005506 0.42 0.001 SDC2 NM_002998 0.380.000 SDPR NM_004657 0.03 0.005 SENP2 AF151697 0.44 0.006 SEPP1NM_001085486 0.00 0.005 SFRP1 NM_003012 0.37 0.000 SHOC2 NM_007373 0.390.000 SIGIRR NM_021805 0.47 0.000 SLC17A5 NM_012434 0.14 0.001 SLC22A5NM_003060 0.21 0.001 SLC9A3R2 NM_004785 0.29 0.000 SMPD1 NM_000543 0.170.000 STAT1 NM_139266 0.19 0.000 STAT6 NM_003153 0.00 0.000 STSNM_000351 0.10 0.007 STYK1 NM_018423 0.05 0.013 SUMF1 NM_182760 0.280.000 TAGLN NM_001001522 0.01 0.000 TFAP2A NM_003220 0.03 0.005 THBS2NM_003247 0.02 0.000 THRA NM_199334 0.31 0.000 THRB NM_000461 0.10 0.014TNXB NM_019105 0.26 0.043 TPM2 NM_213674 0.12 0.000 TRIOBP NM_0070320.15 0.003 TRIP11 NM_004239 0.45 0.001 TSC22D3 NM_004089 0.14 0.000TWIST1 NM_000474 0.01 0.003 VCAN NM_004385 0.04 0.000 VCL NM_014000 0.280.000 VLDLR NM_003383 0.15 0.000 WISP1 NM_003882 0.05 0.013 WNT5ANM_003392 0.01 0.000 YAP1 NM_006106 0.41 0.007 ZBTB7B NM_015872 0.440.000

TABLE X5 Up-regulated Neural Precursor genes (NSLC vs. hNPC) AccessionFold change of NSLC GeneSymbol Number compared to hNPC² p-value ACTL6ANM_178042 2.33 0.000 BCAT1 NM_005504 9.92 0.000 BDNF NM_170735 33.900.000 BMP2 NM_001200 17.71 0.000 CDKN2A NM_058197 5.57 0.000 COL18A1NM_030582 7.22 0.001 DIAPH1 NM_005219 2.33 0.001 EDNRB NM_003991 2.780.000 IDE NM_004969 2.74 0.000 LIMK1 NM_002314 3.61 0.000 MAPK8IP1NM_005456 2.77 0.000 MCHR1 NM_005297 4.02 0.000 MYLIP NM_013262 4.220.000 NEDD4 NM_006154 2.23 0.000 NOS2A NM_000625 267.58 0.000 PCSK9NM_174936 9.65 0.000 PSEN2 NM_000447 2.07 0.000 SMAD1 NM_005900 3.090.000 TBX1 NM_080647 3.65 0.028 TGFB1 NM_000660 6.66 0.000 ²Fold changerepresents the up-regulation of the gene in the NSLC samples as comparedto the hNPC samples. (n = 3 for hNPC samples, n = 3 for NSLC samples).

TABLE X6 Up-regulated Glia genes (NSLC vs. hNPC) Accession Fold changeof NSLC GeneSymbol Number compared to hNPC¹ p-value ACSL4 NM_004458 2.100.000 BDNF NM_170735 33.90 0.000 BMP4 NM_001202 20.55 0.001 CP NM_000096159.46 0.000 CSPG4 NM_001897 4.94 0.000 FOXC1 NM_001453 5.12 0.000 GFAPNM_002055 13.67 0.000 GJB2 NM_004004 7.25 0.000 GLIPR1 NM_006851 5.580.000 ITGA3 NM_002204 24.64 0.000 LMO3 NM_018640 129.25 0.000 NEFLNM_006158 7.14 0.000 NKX6-2 NM_177400 11.50 0.000 NRTN NM_004558 3.390.001 PDCD11 NM_014976 2.48 0.000 PLP1 NM_000533 20.64 0.000 TGFB1NM_000660 6.66 0.000 TSPAN12 NM_012338 2.58 0.006

TABLE X7 Up-regulated Regenerative genes (NSLC vs. hNPC) Accession Foldchange of NSLC GeneSymbol Number compared to hNPC¹ p-value ATR NM_0011842.57 0.000 BMP2 NM_001200 17.71 0.000 BMP4 NM_001202 20.55 0.001 CAV3NM_001234 26.23 0.000 CCND1 NM_053056 10.34 0.000 CDKN2A NM_058197 5.570.000 CEBPB NM_005194 2.58 0.000 GAL NM_015973 12.21 0.000 GAP43NM_002045 4.27 0.000 HOXB4 NM_024015 133.37 0.000 SMAD3 NM_005902 2.270.000

In order to investigate the differentiation potential of NSLCs toneuronal lineages (Neurons, astrocytes, and oligodendocytes),neurospheres were dissociated and plated in laminin/poly-D-Lysine (10μg/ml; Sigma) in differentiation medium for two weeks. Thedifferentiation towards neuronal lineage was performed using twodifferent mediums: NbActive medium (BrainBits™) supplemented with BrainDerived Neurotrophin Factor (BDNF, 20 ng/ml, Peprotech),all-trans-retinoic acid (ATRA, 5 μM, Spectrum), and bFGF (40 ng/ml,Peprotech) or NeuroCult™ differentiation medium (NeuroCult™Differentiation kit, StemCell Technologies), supplemented with BDNF (20ng/ml, Peprotech) and bFGF (40 ng/ml, Peprotech). After two weeks inculture, the cells were stained with the neuronal marker βIII-tubulin,astrocyte markers GFAP and S100β, andoligodendrocyte marker CNPase. Thecells were fixed with 4% formaldehyde and the primary antibodies wereadded in 5% normal goat serum/PBS as follows: Mouse antibodyβIII-tubulin (1:200, Abcam), rabbit antibody S100β (1:100, Abcam), andChicken antibody CNPase (1:50, Abcam). Secondary antibodies are added in5% normal goat serum/PBS as follows: Goat anti mouse Alexa546™ (1:200,Invitrogen), Goat anti rabbit Alexa488™ (1:200, Invitrogen), and Goatanti-chicken cy5 (1:100, Jackson ImmunoResearch Labs).

Immunohistochemistry analysis showed that NbActive medium promoted thedifferentiation equally to neuronal (48.66±14.07%, βIII-tubulin) andpotential early oligodendrocyte lineages (50.01±4.04%, CNPase) and to alower percentage of astrocyte cells (2.68±1.13%, S100β), while NS-Adifferentiation medium induced the differentiation mainly to neurons(64.89±4.11%, βIII-tubulin) and astrocytes (35.94±4.04%, S100beta), anda low percentage of potential early oligodendrocytes cells (8.68±2.71%,CNPase). The NSC-A medium was selected over NbActive for furtherdifferentiation studies. Differentiation of cells in NS-Adifferentiation medium promote the differentiation of hNPC and NSLCsimilarly as shown in Table 17 by the decrease of the percentage ofsox2, musashi and nestin positive cells. NSLCs were differentiated toneuronal (74.3±0.1, GABA), astrocyte lineage (65.6±0.0, S100beta) and toa lower percentage of oligodendrocyte cells (5.2±0.6, CNPase). The samepattern of tripotent lineage differentiation was observed with hNPCs(Table 17).

TABLE 17 The percentage of cells stained positive for neural stem celland neuronal lineage markers in transfected and untransfected cells.NSLCs and hNPCs were cultured in NS-A- differentiation mediumsupplemented with BDNF (20 ng/ml) and FGF (40 ng/ml), cultures wereincubated at 37° C., 5% CO₂, 5% O₂ for three weeks. The percentage ofimmunopositive cells was determined by Cellomics ™ and represented asmean ± SD (n = 5). Sox2 Nestin Musashi S100 O4 GABA Tripotent hNPC 73.8± 0.5 46.1 ± 5.2 22.1 ± 7.0 20.8 ± 1.3 6.4 ± 2.9 68.5 ± 1.6 medium NSLC68.6 ± 3.9 41.0 ± 5.4 26.7 ± 5.0 65.6 ± 0.0 8.2 ± 0.6 74.3 ± 0.1

Several additional antibodies to neuronal antigens were used tocharacterize, in more detail, the nature of differentiated cells.Antibodies against microtubule-associated protein (MAP2b), NCAM, andsynaptophysin were used as recommended by the antibody manufacturer.After three weeks in differentiation medium, there was adifferentiation-induced reduction in markers of precursors cells and anincrease in mature neuronal markers. The percentage of neural precursormarkers such as Sox2 were decreased during differentiation, whilep75NTR, βIII-tubulin and GABA were increased with lengtheningdifferentiation time (FIG. 6); however, 04 positive cells were very lowafter 3 weeks of differentiation of hNPCs (6.4±2.9) and NSLCs (8.2±0.6).Synaptophysin, an antibody used to identify functional neuronal cells,was increased following 2 and 3 weeks of differentiation, indicatingmaturity of the neuronal cells. GABA and acetycholine markers wereincreased following 2 weeks of differentiation and decreased at week 3.

The morphological changes and expression of a number of neuronalantigens and genes show that the above method results in normal andviable neuronal cells. Additionally, the newly formed neuronal cellshave the morphological criteria of neurons. In addition to the abovemarkers, the differentiated cells were evaluated by characterizingmorphological markers of neurite differentiation. Neuron type cells(cells strongly expressing βIII-tubulin) showed neurite formation afterdifferentiation, including an increase in the average number of neuritesper neuron (from e.g. 1.38±0.1) The same pattern was observed inβIII-tubulin positive cells. Accordingly, the average neurite length(118.3±3.5 μm) and the number of branch points (3.28±0.3) per neuronalso increased. The differentiated neuron-like cells developed longneurites that were greater than three cell diameters in length with agrowth cone at the end, expressed neuron-specific genes, and stoppedproliferating after the induction of differentiation.

Further differentiation was performed using an optimised medium thatpromoted the differentiation towards oligodendrocyte lineage. NSLCs andhNPCs were cultured in NS-A differentiation medium as describedpreviously supplemented with FGF-2 (10 ng/ml, Peprotech) and sonichedgehog (SHH, 100 ng/ml, Peprotech) for 4 days. After 4 days medium waschanged to NS-A differentiation medium supplemented by T3 (60 ng/ml,Peprotech), IGF1 (10 ng/ml, Peprotech), NT-3 (10 ng/ml, Peprotech), andPDGF (10 ng/ml, Peprotech). Cells were cultured for 20 days at 37° C.,5% CO₂.

TABLE 18 The percentage of cells stained positive for neural stem celland neuronal lineage markers in transfected and untransfected cells.NSLCs and hNPCs were cultured in differentiation medium supplementedwith SHH (100 ng/ml, Peprotech), T3 (60 ng/ml, Peprotech), IGF1 (10ng/ml, Peprotech), NT-3 (10 ng/ml, Peprotech), and PDGF (10 ng/ml,Peprotech) to induce differentiation towards oligodendrocytes. Thepercentage of immunopositive cells was determined by Cellomics ™ andrepresented as mean ± SD (n = 5). % of positive cells Sox2 NestinMusashi S100 O4 GABA hNPC 84.3 ± 3.7 26.9 ± 4.4 51.8 ± 2.9  33.4 ± 1.940.1 ± 6.4  89.6 ± 0.8 NSLC 69.3 ± 4.4 24.3 ± 2.5 45.1 ± 11.1 51.6 ± 9.58.5 ± 0.6 76.9 ± 1.4

Quantification of the differentiation of hNPCs and NSLCs revealed apopulation of cells that were positively stained for O4. As shown inTable 18, the percentage of O4 positive cells was more pronounced indifferentiated hNPC (40.1±6.4%) as compared to differentiated NSLCs(8.5±0.6%) when using the above differentiation protocol.

This study showed that transfecting the cells with one or two neurogenictranscription factors in the presence of a DNA demethylator or smallmolecules for epigenetic modification achieves stable reprogrammed cells(NSLCs). Like a DNA demethylator, epigenetic modification (inhibition ofacetylation and methylation) are sometimes useful in boosting thereprogramming process. These cells possess and retain neural stem cellproperties as determined by: (1) the expression of neural stem cellgenes and proteins, (2) the capacity to generate and grow asneurospheres starting from a single cell, and (3) to differentiate toneuronal lineages in differentiation conditions. When differentiated toneurons, cells display one or more neural-specific morphological,physiological and/or immunological features associated with a neuronalcell type. Useful criteria include morphological features (longprocesses or neurites), physiological, and/or immunological featuressuch as expression of a set of neuronal-specific markers or antigens.Furthermore, NSLCs readily turn into a tripotent-like precursor cellwith differentiation potential to a high percentage of neuronal,astrocytes and lower percentage of oligodendrocyte populations.

Example VI Implication of BMP Signaling Pathway in the Reprogramming ofHFFs

This study was designed to evaluate the role of Noggin in the process ofde-differentiation of HFFs towards NSLCs. HFFs were cultured and treatedwith cytochalasin B as described in Example III. After two days oftreatment, cells were transfected by Nucleofection as described inExample II with the constructed vector Msi1/Ngn2. Briefly, afterpreparing the cells, they were mixed with 2 μg of total DNA (Msi1/Ngn2)and were co-transfect with MBD2 (2 μg), by the Amaxa's Nucleofector™according to the manufacturer's protocol. The samples were thentransferred into a Laminin (10 μg/ml, Sigma) coated culture plate andcultured in the presence of Neural Proliferation Medium (NeuroCult™proliferation Kit, StemCell Technologies) with recombinant hFGF (20ng/ml, Peprotech), recombinant hEGF (20 ng/ml, Peprotech), and with orwithout the presence of Noggin (20 ng/ml, Peprotech). Samples werecollected at different time points (1, 3, 4, 6, and 8 days) to analyzeneuronal gene expression by RT-PCR and protein expression levels byimmunohistochemistry.

Fluorescent immunohistochemical staining was performed on samples after4 days of transfection as previously described in Example I. Transfectedcells were stained and analyzed for expression of Sox2, the percentageof Sox2 was 33.3±1.00% in the presence of Noggin compared to 27.5±0.50%without the presence of noggin at day 4. RT-PCR analysis of relativeexpression of neuronal precursor cell markers such as nestin and Sox2after transfection of HFFs with pCMV-Msi1-2A-Ngn2 and pCMV6-XL5-MBD2with or without the presence of Noggin (20 ng/ml) was associated with anincrease in nestin and Sox2 starting at day 3 and maintained until day 8(Table 19). No difference in the expression was noticed in the absenceof Noggin. Inhibiting the BMP signaling pathway by Noggin thus enhancedreprogramming, but had no reprogramming effect on its own.

TABLE 19 RT-PCR analysis of relative expression of neuronal precursorcell markers such as nestin and Sox2 after transfection of HFF withpCMV-Msi1-2A-Ngn2 and pCMV6-XL5-MBD2 with or without Noggin (20 ng/ml).Relative expression of Sox2, and nestin was increased after transfectionwith and without Noggin. ACHE GFAP NES SOX2 TUBB3 Rel. Std. Rel. Std.Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.Exp. Dev. #1 Msi1/Ngn2 + 7.08 1.70 2.97 0.42 1.33 0.10 0.93 0.91 1.370.10 MBD2/+Noggin Day 1 #2 Msi1/Ngn2 + 7.34 1.03 2.01 0.08 1.28 0.180.60 0.10 0.98 0.05 MBD2/+Noggin Day 2 #3 Msi1/Ngn2 + 9.67 2.41 15.131.66 1.98 0.20 6333.63 277.87 0.95 0.07 MBD2/+Noggin Day 3 #4Msi1/Ngn2 + 11.68 2.65 194.07 25.22 4.19 0.52 20231.33 1034.29 1.90 0.45MBD2/+Noggin Day 4 #5 Msi1/Ngn2 + 3.58 0.66 227.99 16.83 1.68 0.096298.51 289.84 0.96 0.17 MBD2/+Noggin Day 6 #6 Msi1/Ngn2 + 10.89 0.57650.34 22.92 4.42 0.03 18134.90 63.93 1.81 0.06 MBD2/+Noggin Day 8 #7Ctrl 1.01 0.19 1.00 0.05 1.00 0.02 1.12 0.70 1.00 0.09 Untransfected +Noggin Day1 #8 Msi1/Ngn2 + 2.79 0.83 1.62 0.19 0.99 0.08 1.28 0.25 0.750.01 MBD2/−Noggin Day 1 #9 Msi1/Ngn2 + 3.79 0.91 1.47 0.08 1.23 0.081.36 0.08 0.72 0.07 MBD2/−Noggin Day 2 #10 Msi1/Ngn2 + 6.18 0.59 14.601.85 2.62 0.30 10949.28 448.28 0.90 0.01 MBD2/−Noggin Day 3 #11Msi1/Ngn2 + 5.63 0.74 74.56 16.56 2.97 0.21 19623.99 3109.69 0.75 0.11MBD2/−Noggin Day 4 #12 Msi1/Ngn2 + 3.21 0.96 232.42 5.47 1.47 0.0715311.64 1909.23 0.86 0.03 MBD2/−Noggin Day 6 #13 Msi1/Ngn2 + 3.82 0.52496.99 75.81 3.32 0.32 26892.31 1817.05 2.05 0.10 MBD2/−Noggin Day 8 #14Ctrl 1.08 0.57 1.01 0.14 1.00 0.04 1.15 0.81 1.00 0.00 Untransfected−Noggin Day1

Example VII NSLCs Created from HFF Cells are not Skin-Derived Precursors(SKPs)

It's known that cells termed skin-derived precursors (SKPs) may residein adult human skin (Fernandes et al., 2004). These cells are capable ofproliferating in response to EGF and bFGF and express nestin, versicanand fibronectin, and can differentiate into both neuronal and mesodermalprogeny. In order to verify that NSLCs are distinct from SKPs,differentiation towards adipocyte cells was performed. Adipose derivedstem cells (ADSC) were maintained in StemPro™ MSC serum free medium(Invitrogen) on flasks coated with CellStart™ (Invitrogen). CellStart™was diluted 1:100 in dPBS/Ca²⁺/Mg²⁺ and the flask incubated for 2 hoursat 37° C. Cells are passaged every 3 to 4 days using Accutase™ andmedium was changed every 2 days. Three to four days before initiatingdifferentiation, ADSCs and NSLCs were seeded in 6-wellplates inCellStart™ (1:100 in dPBS/Ca²⁺/Mg²⁺/2 hours at 37° C.) coated tissueculture plates. When cells reached confluence (after 3 to 4 days),proliferation media were replaced by differentiation medium consistingin DMEM/F12 (50:50), ITS (1:100), HEPES (1:100), GlutaMAX™ (1:100), T3(0.2 nM), Rosiglitasone (0.5 μg/ml), IBMX (100 μM) and Dexamethasone (1μM). Three days after, IBMX and dexamethasone were withdrawn from thedifferentiation medium. At day 10, cells were fixed with a 4%formaldehyde solution for 10 min and stained with Oil Red O (Invitrogen)staining solution for 15 min. Staining was removed and cells washedtwice with PBS. Adipose cells appeared red with lipid dropletsspecifically stained with Oil Red O, however NSLCs were stainednegative, with no presence of lipid droplets in the cells, and the cellsadopted neuronal cell morphology.

Immunohistochemistry analysis confirmed that NSLCs are distinct fromSKPs (FIG. 24): NSLCs stained positive for p75NTR and negative forfibronectin and versican, while SKPs express fibronectin and versicanand do not express p75NTR (Fernandes et al., 2004). This study indicatesthat NSLCs represent a tripotent-like precursor cell and they are not asubpopulation of SKPs.

Example VIII BDNF Release from Neural-Like Cells (NLCs)

Neural Stem-Like Cells (NSLCs) differentiated into neuronal and glialcells were kept in culture for 55 days, and BDNF released in theconditioned medium was measured by antigen-capture ELISA at differenttime points and compared to the release in mature neurons (ScienCell),undifferentiated Neural Human Normal Precursor cells (NHNP, Lonza) aswell as to undifferentiated NSLCs and untransfected cells (HFF).Conditioned medium from each group was collected, centrifuged, and thenstored at −80° C. until assaying. BDNF concentrations were measured byELISA kits (BDNF E_(max) Immunoassay System, Promega Corporation, USA),according to the manufacturer's instructions. Briefly, 96-well ELISAimmunoplates were coated with Anti-BDNF (CatNb#G700B) diluted 1/1000 incarbonate buffer (pH 9.7) and incubated at 4° C. overnight. Thefollowing day, all wells were washed with TBS-Tween™ 0.5% beforeincubation with Block/Sample buffer 1× at room temperature for one hourwithout shaking. After blocking, standards and samples were added to theplates and incubated and shaken (450±100 rpm) for 2 h at roomtemperature. Subsequently, after washing with TBS-Tween™ wash buffer,plates were incubated for 2 h with Anti-Human BDNF pAb (1:500 dilutionin Block & Sample 1× Buffer) at 4° C. After incubation, plates werewashed five times with TBS-Tween™ 0.5% wash buffer and 100 μl of dilutedAnti-IgYHRP Conjugate was added to each well (1:200 dilution in Block &Sample 1× Buffer) and incubated for 1 hour at room temperature withshaking (450±100 rpm). Then, plates were washed five times withTBS-Tween™ 0.5% wash buffer and 100 μl of TMB One Solution was added toeach well. Following 10 minutes incubation at room temperature withshaking (450±100 rpm) for the BDNF plate, a blue color formed in thewells. After stopping the reaction by adding 100 μl of 1N hydrochloricacid, the absorbance was read at 450 nm on a microplate reader (Synergy4™) within 30 minutes of stopping the reactions. Concentration ofreleased BDNF in the supernatants was determined according to thestandard curves.

ELISA results revealed that BDNF was released at the same concentrationfrom differentiated Neuron-Like Cells (NLCs differentiated from NSLCs)and normal Human neuron cells starting at day 11 and remained until day55 (Table 20), while no BDNF (except for tiny amounts in theuntransfected HFF group) was released in the other groups.

TABLE 20 Quantification of BDNF release by Neural-Like Cells (NLCs) thathad been differentiated for 55 days from Neural Stem-Like Cells (NSLCs)that had been created from transfected HFFs. BDNF release from NLCs intothe medium, at different time points, was measured by antigen-captureELISA and compared to BDNF release of normal mature human neurons(ScienCell). Control medium Neurons NLC day 0 day 11 1.55 30.25 22.99day 18 0.33 29.49 25.15 day 24 0.33 22.01 26.39 day 34 0.23 25.53 32.21day 41 0.27 19.02 22.43 day 55 0.02 20.73 30.01

In addition to adopting neuronal morphology criteria, the NLCs werefunctional and possessed the capacity to release neurotrophic factor(BDNF). Generating reprogrammed neuronal-like cell lines that canlocally deliver these neurotrophic factors could be used as a method totreat several neurological conditions and may offer crucial benefits inregeneration and functional recovery from brain and other injuries.

Example IX Reprogramming of Different Cell Types Towards NSLCs

This study was performed to investigate the capacity of keratinocytes(Invitrogen), human Adipocytes Derived Stem Cells (ADSCs, Invitrogen)and human hematopoietic stem cells (CD34⁺, Invitrogen) cells into neuralstem-like cells.

Preparation of Human CD34⁺ Cells, Human ADSC and Human Keratinocytes

Human mobilized peripheral blood CD34⁺ cells were purchased fromStemCell Technologies and expanded as a floating culture in Petri Dishesin complete StemPro™®-34 Serum-free Medium (Invitrogen) supplementedwith Stem Cell Factor (SCF, 150 g/ml, Peprotech), GranulocyteColony-Stimulating Factor (GM-CSF, 37.5 ng/ml, Peprotech) and IL-3 (75ng/ml, Peprotech). Medium supplemented with cytokines was changedeveryday 2-3 days after centrifugation of the cell suspension at 300×gfor 10 min. Every other day the cytokines were added directly to theculture without changing the media. Cells were incubated at 37° C., 5%CO₂. For their passaging, cells were centrifugated, resuspended in theabove medium plus cytokines and placed into the adequate number of Petridishes.

Human Adipose-Derived Stem Cells (ADSC) were purchased from Invitrogenand expanded in complete StemPro™ MSC Serum-free medium (Invitrogen) onCellStart™™ (Invitrogen) coated flasks (diluted 1:100 in PBS containingCa²⁺/Mg²⁺) at a cell density of 1×10⁴ cells/cm². Medium was replacedevery two days with fresh pre-warmed complete StemPro™ MSC SFM. Cellswere incubated at 37° C., 5% CO₂. Cells were sub-passaged when 80%confluent by incubation for 3-5 min in pre-warmed TrypLE™™ (Invitrogen)and then collected in StemPro™ MSC medium. After centrifugation at 1500rpm for 5 min, cells were seeded on CellStart™™ coated flasks asdescribed above.

Primary human keratinocytes were purchased from Invitrogen and expandedin Defined Keratinocyte Serum-free medium on Coating matrix (Invitrogen)coated flasks (Invitrogen) at a cell density of 5×10³ cells/cm². Thecells were incubated at 37° C., 5% CO₂. Media was replaced with fresh,complete growth media every two to three days until subculture. Once thecells had reached 70-80% confluency, media was removed and the cellswere incubated in Versene™ (Invitrogen) for 3-5 min at room temperature.Versene™ was removed, and pre-warmed 0.05% trypsin-EDTA (Invitrogen) wasadded to the flasks. After 5-10 min incubation, growth medium containingSoybean Trypsin inhibitor (Invitrogen) was added to the flasks and thecells gently triturated. After centrifugation at 100×g for 10 min, cellswere resuspended in the desired volume of pre-warmed, complete growthmedium on coated flasks as described above.

Prior to transfection, cells were trypsinized and transientlyco-transfected with pCMV-Msi1-Ngn2 and pCMV6-XL5-MBD2 as previouslydescribed in Example IV using the Shuttle and plated into a cultureplate coated with laminin (Sigma, 10 μg/ml). Starting one day aftertransfection, cells were treated with VPA (1 mM) for 4 days and themedium was changed gradually to proliferation medium supplemented withFGF (20 ng/ml) and EGF (20 ng/ml) and were cultured for 18 days at 37°C., 5% CO₂ and 5% O₂. Cells were then analyzed for neural stem cellmarkers by RT-PCR and Immunohistochemistry.

Further analysis and quantification of the reprogrammed cells revealed apopulation of NSLCs engendered from keratinocyte and CD34⁺ cells. RT-PCRAnalysis revealed an increase of relative expression of neural stem cellmarkers such as Sox2, nestin, GFAP, and βIII-tubulin after transfectingKeratinocyte and CD34⁺ by Msi1 and Ngn2. Relative expression of nestinand GFAP was enhanced in NSLCs created from keratinocytes and CD34⁺cells as compared to NSLCs from HFFs; however, the reverse was true forSox2 and ACHE expression. βIII-tubulin (TUBB3) and Map2b expression washighest in NSLCs created from CD34⁺ cells, followed by NSLCs createdfrom HFF (Table 21). This data shows that different types of NSLCs withdifferent gene expression profiles (and characteristics) can be createdfrom different types of starting/source cells (and the same has beenobserved for creating some other types of stem-like cells discussed inthis application). The data is also intriguing since it was not expectedthat keratinocytes (which are derived from the ectoderm just asendogenous neural stem cells) would have a lower expression than HFFsfor all the genes analyzed except for Nestin (it was expected thatkeratinocytes would be the easiest to reprogram into NSLCs since theyare derived from the ectoderm).

TABLE 21 RT-PCR analysis was performed after one month of transfectionof human fibroblasts (HFF), Keratinocytes, and CD34⁺ cells withMsi1/Ngn2 (MSI1/NGN2), in the presence MBD2 with VPA treatment. Cellswere cultured on coated culture plates in proliferation medium (StemCellTechnologies) supplemented with EGF (20 ng/ml) and FGF (20 ng/ml) for 18days. Untransfected cells were considered as negative control. NES MAP2TUBB3 ACHE GFAP SOX2 Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std.Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. #1Day12 1.00 0.07 1.00 0.05 1.00 0.01 1.01 0.15 1.00 0.02 1.08 0.59Untransfected HFF #2 Day12 HFF 2.25 0.03 21.48 2.09 3.41 0.45 12.92 1.88558.69 80.08 71513.12 14146.80 Msi1/Ngn2 + MBD2 #3 Day18 HFF 2.56 0.1517.12 0.14 2.65 0.02 4.13 0.64 75.96 8.82 84794.40 318.54 Msi1/Ngn2 +MBD2 #4 1.07 0.54 1.00 0.07 1.00 0.02 1.01 0.19 1.06 0.48 1.00 0.01Untransfected Keratinocytes #5 Day 12 11452.65 1137.13 0.96 0.11 6.780.28 1.09 0.05 5815.54 510.91 975.81 7.47 Keratinocytes Msi1/Ngn2 + MBD2#6 Day 18 12593.79 431.06 0.93 0.04 6.41 0.27 0.48 0.03 1295.15 32.051047.17 139.48 Keratinocytes Msi1/Ngn2 + MBD2 #7 1.00 0.04 1.01 0.161.00 0.00 1.00 0.01 1.10 0.66 1.01 0.21 Untransfected CD34+ #8 Day 18839.57 134.51 346.61 33.97 33.91 4.38 0.28 0.00 2790.18 304.43 25080.3535.93 CD34+ Msi1/Ngn2 + MBD2 hNPC 4.56 0.07 278.36 11.50 0.81 0.06 72.651.83 1285.73 5.27 565552.30 41717.72

Immunohistochemistry revealed positive staining for GFAP, Sox2, andnestin as shown in FIG. 7. NSLCs developed from HFF yield a higherpercentage of positive staining for Sox2 and GFAP (55.8±3.8 and78.1±2.4) as compared to CD34⁺ cells (42.8±2.7 and 24.2±4.4), andkeratinocytes (47.1±2.1 and 43.4±8.9). The percentage of nestin positivecells was high in Keratinocytes (77.6±10.7) and HFF (68.45±12.9) andlower in CD34⁺ cells (15.5±2.7) (Table 22). Sox2 and Nestin positivestaining was undetectable in ADSCs.

TABLE 22 The percentage of Sox2 and nestin positive cells for neuralstem cell markers after transfecting fibroblast, keratinocyte, and CD34⁺cells with pCMV-Msi1-Ngn2 in the presence of MBD2 and VPA. Cells werecultured on coated culture plates in proliferation medium (StemCellTechnologies) supplemented with EGF (20 ng/ml) and FGF (20 ng/ml) for 18days. Untransfected cells were considered as negative control. Thepercentage of immunopositive cells was determined by Cellomics ™ andrepresented as mean ± SD (n = 5). % positive Untransfected cells cellsFibroblasts Keratinocytes CD34+ Sox2 1.5 ± 1.7 55.8 ± 3.8 47.1 ± 2.142.8 ± 2.7 GFAP 0.04 +/− 0.2 78.1 ± 2.4 43.4 ± 8.9 24.2 ± 4.4 Nestin 0.3 +/− 0.3 68.45 ± 12.9  77.6 ± 10.7 15.5 ± 2.7

NSLCs generated from keratinocytes and CD34⁺ cells were tested fortripotent capacity. Further differentiation studies were performed toinduce differentiation of these NSLCs towards neuronal lineage, usingNeuroCult™ differentiation medium (NeuroCult™ differentiation Kit,StemCell Technologies) supplemented with BDNF (20 ng/ml, Peprotech) andbFGF (40 ng/ml, Peprotech) as described in Example V. NSLCs generatedfrom HFFs and hNPCs were used as controls, cultures were incubated at37° C., 5% CO₂, 5% O₂ for three weeks. Samples were collected or fixedat Day 14 and 28 following differentiation for further analysis. RT-PCRanalysis revealed decrease of undifferentiated genes (Nestin and Sox2)and increased of differentiated genes (Map2, βIII-tubulin, CNPase, andGFAP) as shown in Tables 23A, 23B, 23C and 23D.

TABLE 23A RT-PCR analysis was performed on NSLCs generated from humanfibroblasts (HFF), keratinocytes, and CD34⁺ cells that were cultured onPoly-D-Lysin/Laminin coated culture plates in differentiation medium for28 days (StemCell Technologies) supplemented with BDNF (20 ng/ml) andFGF (40 ng/ml). hNPCs (Lonza) were considered as a positive control.hNPCs had a much lower increase in ACHE, GFAP, and MAP2b (which actuallydecreased in hNPCs), but an increase in Nestin, compared to NSLCs underdifferentiation conditions. NES MAP2 TUBB3 ACHE GFAP SOX2 SOX9 CNP Rel.Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel.Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp.Dev. Exp. Dev. hNPC Control 1.00 0.08 1.00 0.10 1.00 0.08 1.01 0.16 1.000.09 1.01 0.16 1.00 0.12 1.00 0.09 Diff. hNPC Day 3.86 0.20 0.65 0.054.87 0.57 0.74 0.52 97.26 7.13 1.85 0.21 0.50 0.04 1.43 0.05 14 Diff.hNPC Day 1.86 0.06 0.68 0.02 3.67 0.13 1.33 0.09 102.74 1.89 1.29 0.010.73 0.05 1.37 0.02 28 NSLC Control 1.00 0.04 1.00 0.04 1.00 0.04 1.000.03 1.00 0.01 1.00 0.01 1.00 0.02 1.00 0.05 Diff. NSLC Day 1.38 0.011.00 0.09 2.06 0.02 1.57 0.24 1.79 0.12 0.73 0.01 0.56 0.01 1.31 0.05 14Diff. NSLC Day 0.62 0.02 0.90 0.08 5.14 0.21 6.47 0.78 5.70 0.15 1.300.02 0.79 0.03 1.41 0.01 28 HFF-NS 1.00 0.00 1.00 0.05 1.00 0.01 1.000.07 1.00 0.00 1.00 0.07 1.00 0.01 1.00 0.02 Control Diff. HFF-NS 2.700.08 3.08 0.12 3.24 0.14 59.93 5.85 478.97 0.27 2.90 0.32 0.81 0.03 4.020.35 Day 14 Diff. HFF-NS 1.27 0.05 1.48 0.11 1.59 0.03 24.62 1.00 576.8020.98 1.52 0.00 0.86 0.08 2.74 0.23 Day 28 Kerat-NS 1.00 0.06 1.00 0.021.00 0.03 1.00 0.11 1.00 0.01 1.00 0.07 1.00 0.02 1.00 0.01 ControlDiff. Kerat-NS 2.43 0.06 3.48 0.08 2.82 0.11 56.22 5.58 665.91 10.523.09 0.29 1.01 0.14 3.72 0.17 Day 14 Diff. Kerat-NS 0.81 0.03 1.72 0.001.61 0.18 26.09 1.12 673.65 11.34 1.29 0.03 1.12 0.03 2.02 0.05 Day 28CD34+-NS 1.00 0.05 1.00 0.07 1.00 0.04 1.00 0.08 1.00 0.00 1.00 0.081.00 0.02 1.00 0.07 Control Diff. CD34+-NS 2.21 0.04 3.47 0.07 2.75 0.0457.87 6.68 407.54 52.07 2.90 0.18 1.10 0.05 3.54 0.02 Day 14 Diff.CD34+-NS 0.79 0.04 1.48 0.01 1.83 0.37 26.92 3.73 485.51 10.66 1.02 0.041.20 0.09 2.34 0.05 Day 28

TABLE 23B RT-PCR analysis was performed on undifferentiated NSLCsgenerated from human fibroblasts (HFF), keratinocytes, and CD34⁺ cellsthat were cultured on Laminin coated culture plates in Proliferationmedium for 4 days (StemCell Technologies) supplemented with EGF (20ng/ml) and FGF (20 ng/ml). Relative expression calibrated toundifferentiated hNPCs. NES MAP2 TUBB3 ACHE GFAP SOX2 SOX9 CNP Rel. Std.Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std.Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.Exp. Dev. Undifferentiated 1.00 0.08 1.00 0.10 1.00 0.08 1.01 0.16 1.000.09 1.01 0.16 1.00 0.12 1.00 0.09 hNPC Control Day 4 Undifferentiated1.23 0.05 0.12 0.00 1.12 0.04 0.09 0.00 21.45 0.26 0.65 0.01 0.28 0.010.37 0.02 NSLC Control Day 4 Undifferentiated 0.94 0.00 0.12 0.01 0.920.01 0.03 0.00 0.38 0.00 0.37 0.02 0.32 0.00 0.31 0.00 HFF-NS ControlDay 4 Undifferentiated 1.00 0.06 0.09 0.00 0.97 0.03 0.03 0.00 0.23 0.000.38 0.03 0.26 0.00 0.30 0.00 Kerat-NS Control Day 4 Undifferentiated1.10 0.05 0.12 0.01 0.95 0.04 0.04 0.00 0.33 0.00 0.44 0.04 0.26 0.000.30 0.02 CD34+-NS Control Day 4

TABLE 23C RT-PCR analysis was performed on differentiated NSLCsgenerated from human fibroblasts (HFF), keratinocytes, and CD34⁺ cellsthat were cultured on Poly-D-Lysin/Laminin coated culture plates indifferentiation medium for 14 days (StemCell Technologies) supplementedBDNF (20 ng/ml) and FGF (40 ng/ml). Relative expression calibrated toDay 14 differentiated hNPCs. NES MAP2 TUBB3 ACHE GFAP SOX2 SOX9 CNP Rel.Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel.Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp.Dev. Exp. Dev. Diff. hNPC Day 1.00 0.05 1.00 0.07 1.00 0.12 1.15 0.801.00 0.07 1.00 0.11 1.00 0.08 1.00 0.03 14 Diff. NSLC Day 0.44 0.00 0.180.02 0.47 0.00 0.22 0.03 0.40 0.03 0.26 0.00 0.31 0.00 0.34 0.01 14Diff. HFF-NS 0.66 0.02 0.56 0.02 0.62 0.03 2.96 0.29 1.86 0.00 0.58 0.060.52 0.02 0.87 0.08 Day 14 Diff. Kerat-NS 0.63 0.02 0.51 0.01 0.56 0.022.78 0.28 1.56 0.02 0.64 0.06 0.54 0.08 0.79 0.04 Day 14 Diff. CD34+-NS0.63 0.01 0.62 0.01 0.54 0.01 3.77 0.43 1.39 0.18 0.69 0.04 0.58 0.030.76 0.00 Day 14

TABLE 23D RT-PCR analysis was performed on differentiated NSLCsgenerated from human fibroblasts (HFF), keratinocytes, and CD34⁺ cellsthat were cultured on Poly D-Lysin/Laminin coated culture plates indifferentiation medium for 28 days (StemCell Technologies) supplementedwith BDNF (20 ng/ml) and FGF (40 ng/ml). Relative expression calibratedto Day 28 differentiated hNPCs. NES MAP2 TUBB3 ACHE GFAP SOX2 SOX9 CNPRel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std.Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.Exp. Dev. Exp. Dev. Diff. hNPC Day 1.00 0.03 1.00 0.02 1.00 0.04 1.000.07 1.00 0.02 1.00 0.01 1.00 0.07 1.00 0.02 28 Diff. NSLC Day 0.41 0.010.15 0.01 1.56 0.06 0.44 0.05 1.19 0.03 0.66 0.01 0.30 0.01 0.38 0.00 28Diff. HFF-NS 0.64 0.03 0.26 0.02 0.40 0.01 0.59 0.02 2.12 0.08 0.43 0.000.38 0.04 0.62 0.05 Day 28 Diff. Kerat-NS 0.44 0.02 0.24 0.00 0.42 0.050.62 0.03 1.50 0.03 0.38 0.01 0.40 0.01 0.44 0.01 Day 28 Diff. CD34+-NS0.47 0.03 0.25 0.00 0.47 0.10 0.85 0.12 1.57 0.03 0.35 0.01 0.43 0.030.52 0.01 Day 28

Fluorescent immunohistochemical staining was performed on samples after14 days and 28 days of differentiation. The expression of Sox2 andNestin was decreased time dependently in differentiated cells (HFF,keratinocyte, and CD34⁺). This decrease was associated with an increaseof differentiated markers at day 28 such as GFAP (68.51±11.87 forHFF-NC, 59.55±9.12 for Keratinocyte NC, and 61.70±1.48 for CD34⁺-NC). Ahigh percentage for βIII-tubulin positive cells was generated fromdifferentiated NSLCs generated from HFF (57.83±4.49) as compared toβIII-tubulin positive cells generated from Keratinocytes (23.27±2.91)and CD34⁺ cells (39.15±7.99) (Table 24)

TABLE 24 The percentage of cells stained positive for neural stem cellmarkers and neuronal lineage markers in hNPCs (Lonza) and transfectedkeratinocytes, HFF, and CD34⁺ cells with pMsi1/Ngn2/MBD2. Transfectedcells (NSLCs) were cultured in Proliferation medium or differentiationmedium for 28 days at 37° C., 5% CO₂, 5% O₂. The percentage ofimmunopositive cells (Sox2, Nestin, GFAP, S100beta, and βIII-tubulin)was determined by Cellomics ™ and represented as mean ± SD (n = 5). %positive Proliferation 14 days 28 days cells conditions differentiationdifferentiation hNPC Sox2 96.23 ± 0.51 59.05 ± 3.01 41.43 ± 6.05 Nestin41.47 ± 0.23 10.77 ± 4.78 16.14 ± 7.41 s100β 37.38 ± 7.85 49.51 ± 2.39n.d. βIII-tubulin  2.34 ± 0.43 11.54 ± 4.03 23.34 ± 4.77 GFAP  1.16 ±0.14 23.42 ± 2.51 48.04 ± 8.30 HFF-NC Sox2 93.28 ± 0.53 79.48 ± 0.5452.06 ± 9.07 Nestin 29.29 ± 4.72  1.15 ± 0.46  2.18 ± 1.96 s100β 13.51 ±0.28 80.75 ± 3.50  79.38 ± 10.62 βIII-tubulin  3.91 ± 0.33  42.16 ±15.07 57.83 ± 4.49 GFAP  8.41 ± 0.73  59.66 ± 11.48  68.51 ± 11.87Keratinocyte-NC Sox2 96.55 ± 1.01 76.93 ± 5.13 63.11 ± 8.54 Nestin 40.10± 8.41  2.67 ± 1.61  3.57 ± 0.48 s100β 13.58 ± 4.97  76.6 ± 9.72  74.75± 11.21 βIII-tubulin  6.42 ± 2.94 20.58 ± 8.34 23.27 ± 2.91 GFAP  9.36 ±0.34 43.43 ± 2.44 59.55 ± 9.12 CD34⁺-NC Sox2 95.49 ± 2.6  81.18 ± 1.2463.46 ± 5.14 Nestin  51.68 ± 14.27 12.64 ± 1.27 8.46 ± 4.6 s100β  30.1 ±1.03 72.40 ± 4.5  79.57 ± 8.52 βIII-tubulin  5.82 ± 2.08  25.04 ± 19.9539.15 ± 7.99 GFAP 13.99 ± 5.48  51.79 ± 13.68 61.70 ± 1.48 n.d. = notdetermined; ± = standard deviation CD34⁺-NC: neuronal cells generatedafter differentiation of NSLCs generated from CD34⁺ cells. Each datapoint represents the analysis of at least 1000 cells from at least 8images.

The % of Sox2 positive cells decreased faster, the % of Nestin positivecells generally decreased slower, and the % of cells expressing one ofthe differentiation markers (S100β, βIII-tubulin, GFAP) generallyincreased slower in hNPCs than in the NSLCs during differentiation. Outof the three types of created NSLC lines, the % of cells expressing oneof the differentiation markers (S100β, βIII-tubulin, GFAP) generallyincreased slowest in NSLCs created from keratinocytes and fastest inNSLCs created from HFFs.

This study indicates that NSLCs can be created from keratinocytes andCD34⁺ blood cells, and these cells share morphology and markerssimilarly to NSLCs generated from HFF. Similarly to hNPCs, NSLCs createdfrom keratinocytes, CD34⁺ cells, and HFFs had a tendency todifferentiate more towards an astrocyte lineage than a neuronal lineage(except NSLCs created from HFFs had an almost similar number ofβIII-tubulin positive and GFAP positive cells) as shown by the highpercentage of GFAP positive cells during differentiation, which wasconfirmed by S100beta staining. However, the proportion of astrocyte andneuronal cells generated from hNPCs was lower in same cultureconditions, indicating that NSLCs generated from HFF, Keratinocytes, andCD34⁺ cells can give rise to a higher number of neuronal and astrocytecells as compared to hNPCs. NSLCs, whether created from HFFs,Keratinocytes or CD34⁺ cells (or potentially even some other cell), aretripotent cells and possess the capacity to differentiate to neurons,astrocytes, and oligodendrocytes similarly to hNPCs. However, RT-PCR andimmunohistochemistry analysis of transfected ADSCs did not reveal anysignificant expression of neural stem cell genes, indicating a need tooptimize conditions for turning ADSCs to NSLCs or to investigate theeffect of others neurogenic factors that could turn these into NSLCs.

Example X Fabrication 3D Extracellular Matrix (CDM)

Fibroblast cells were cultured in DMEM medium in the presence of 10% FCSas described in Example I, followed by seeding onto 12-well platespre-coated with laminin (10 μg/ml) at a concentration of 2×10⁶ cells/mlin defined CDM Medium consisting of a 3:1 ratio of Dulbecco's modifiedEagle medium (DMEM, high glucose (4.5 g/L) with L-glutamine and sodiumpyruvate) and Ham's F-12 medium supplemented with the followingcomponents: EGF (4.2×10⁻¹⁰M), bFGF (2.8×10⁻¹⁰M), ITS (8.6×10⁻⁵M),dexamethasone (1.0×10⁻⁷M), L-ascorbic acid phosphate magnesium saltn-hydrate (3.2×10⁻⁴M), L-3,3′,5-triiodothyronine (2.0×10⁻¹⁰M),ethanolamine (10⁻⁴M), GlutaMAX™ (4×10⁻³M), glutathione (3.3×10⁻⁶M), and1% penicillin/streptomycin/amphotericin B. By culturing the fibroblastcells at hyperconfluent density in this completely chemically definedmedium causes them to enter a high synthetic phase with a slow-down inproliferation, leading to the production of a living tissue equivalent(LTE) consisting of multiple layers of fibroblasts within de novo 3Dextracellular matrix (CDM) that is completely synthesized by thefibroblasts themselves.

Trans-Differentiation and Reprogramming of Cells within CDM

Day 14 CDM samples were treated with cytochalsin B (10 μg/ml,Calbiochem), with the concentration of cytochalsin B reduced from 10μg/ml to 0 μg/ml (none) over 5 days while at the same time switching themedium from CDM Medium to NbActive medium. Samples were cultured foranother 12 days at 37° C., 5% CO₂, and the medium was changed every day.Samples were fixed to perform immunohistochemistry as describedpreviously to detect Neuronal markers. The following antibodies wereused: mouse anti-nestin 647 (1:100, BD) and anti-βIll-tubulin (1:200,Neuromics). No clear morphology change of the cells was observed withinthe CDM and the immunohistochemical analysis failed to detectβIII-tubulin positive cells. Thus, inducing the trans-differentiation ofcells using only cytochalasin B and chemically-defined neural medium wasnot sufficient to reprogram the cells.

Next, Day 6 CDM samples grown in LAS pre-coated plates at 37° C. and 5%CO₂, were exposed simultaneously to cytochalasin B (10 μg/ml) over 5days, histone deacetylation inhibitor (VPA, 4 mM, Calbiochem) andinhibitor of DNA methylation (5-Azacytidine, 5 μM, Sigma). Four dayslater, the medium was changed to differentiation medium consisting of a3:1 ratio of CDM medium without the presence of EGF and NbActive medium(BrainBits™) supplemented with NT-3 (20 ng/ml, Peprotech) and BDNF (20ng/ml, Peprotech). The ratio of the differentiation medium was increasedgradually day after day until reaching 100% of complete differentiationmedium. After two weeks of treatment, cells were fixed forimmunohistochemical analysis to investigate the identity of the cells.FIG. 18 shows immunostained cells with βIII-tubulin at day 7, indicatingthe de-differentiation of fibroblast cells to neurons. However, one weeklater, these trans-differentiated cells reverted back to fibroblastcells and βIII-tubulin expression was lost (FIG. 8). The loss ofmorphology and βIII-tubulin expression after withdrawal of the primingagents indicate that complete conversion to functional and stablereprogrammed cells did not occur.

Next CDM was treated with VPA (4 mM), 5-Aza (5 μM) and cytochalasin B(10 μg/ml) as above. After 2 days of chemical treatment, fibroblastcells within the CDM were transfected with DNA using Lipofectaminereagent (Invitrogen) as per the manufacturer's protocol. 15 μg of theeukaryotic DNA expression vectors pCMV6-XL5-Pax6, pCMV6-XL5-Msi1 andpCMV6-XL4-Ngn2 (Origene) were used to transfect the cells. 24 hourslater, the media was changed to Neural Progenitor Basal Medium (Lonza)supplemented with Noggin (50 ng/ml), EGF (20 ng/ml), and bFGF (20ng/ml), and the cells were cultured at 37° C., 5% CO₂ and 5% O₂, and themedium was changed every day. At day 6, differentiation was initiated byadding gradually NBActive medium (BrainBits™) supplemented with NT-3 (20ng/ml, Peprotech), all-trans-retinoic acid (ATRA, 5 μM, Spectrum), BDNF(20 ng/ml, Peprotech), and bFGF (40 ng/ml, Peprotech). To characterizethe reprogrammed cells, immunohistochemical analysis and RT-PCR wasperformed at various time points according to the methods described inExample II using primers for nestin, βIII-tubulin, GFAP, MAP2b, andACHE. In agreement with previous studies, un-transfected cells and cellstransfected with Pax6 did not expressed genes specific for neuronallineages (Table 25). On the other hand, following transfection withMsi1, levels of nestin and ACHE were increased to 4-fold and 8-fold,respectively, and this expression was maintained over the 12-day period.Also levels of GFAP mRNA was enhanced time dependently by approximately14 times. Likewise, the same pattern was observed in Ngn2 transfectedcells. While expression of βIII-tubulin and MAP2b were modestlyincreased following transfection with one neurogenic transcriptionfactors the regulation of gene expression after transfecting the cellswith two neurogenic factors, Msi1 or Ngn2 with Pax6, did not furtherincrease the expression of neuronal genes. FIG. 19 shows that expressionof these genes was enhanced when the cells were transfected with Msi1and Ngn2, with βIII-tubulin enhanced to almost 6-fold at day 12.

TABLE 25 RT-PCR analysis of relative expression of neuronal precursorcell markers such as nestin, βIII-tubulin, MAP2b, ACHE, and GFAP aftertransfection of fibroblast cells with pCMV6-XL5-Msi1, pCMV6-XL4-Ngn2,pCMV6-XL5-Pax6, and pCMV6-XL5-MBD2. After 24 h following transfection,CDM I Medium was changed and cells were cultured in proliferation medium(NPBM, Lonza) supplemented with EGF (20 ng/ml. Peprotech) and bFGF (20ng/ml, Peprotech) for one week. Differentiation was induced by changingthe medium to NbActive (BrainBits ™) supplemented with NT-3 (20 ng/ml),bFGF (20 ng/ml), ATRA (5 μM) and Forskolin (10 μM). Cells were incubatedat 37° C., 5% CO₂, 5% O₂ for 12 days. Relative expression of Msi1, Ngn2,Pax6, nestin, βIII-tubulin, ACHE, MAP2b and GFAP in NSLCs and NLCs wasincreased after transfection with both transcription factors Ngn2 andMsi1 with MBD2 as the DNA demethylator. COL5A2 FBN2 NES MAP2 TUBB3 SOX2ACHE GFAP Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Std. Std.Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Rel. Exp. Dev.Rel. Exp. Dev. Rel. Exp. Dev. #1, +CytoB, 1.00 0.07 1.00 0.01 1.00 0.041.00 0.05 1.00 0.05 1.00 0.05 1.00 0.10 1.00 0.11 Control #2, −CytoB,1.00 0.03 1.00 0.08 1.00 0.00 1.00 0.09 1.00 0.09 1.15 0.80 1.01 0.181.00 0.01 Control #3, +CytoB, 0.85 0.04 0.75 0.02 0.60 0.01 0.29 0.010.44 0.00 22.39 5.26 0.81 0.19 10.14 0.15 Msi1, GAD45b #4, −CytoB, 0.870.03 1.81 0.09 1.84 0.04 2.31 0.00 2.09 0.03 20.28 5.33 1.99 0.74 6.030.05 Msi1, GAD45b #5, +CytoB, 0.84 0.04 0.77 0.03 0.44 0.00 0.24 0.000.36 0.01 470.84 13.43 0.63 0.05 103.22 0.80 Ngn2, GAD45b #6, −CytoB,0.75 0.07 1.97 0.02 1.83 0.00 4.40 0.16 2.02 0.10 789.33 60.35 1.70 0.13110.48 4.90 Ngn2, GAD45b #7, +CytoB, 0.74 0.12 1.08 0.00 0.89 0.01 0.510.00 0.63 0.04 1.64 0.98 0.86 0.12 2.49 0.21 Pax6, GAD45b #8, −CytoB,0.66 0.04 2.41 0.09 2.70 0.03 4.96 0.30 3.48 0.07 0.46 0.33 2.97 1.040.43 0.09 Pax6, GAD45b #9, +CytoB, 0.14 0.01 0.28 0.01 1.30 0.03 4.070.11 0.84 0.00 54768.27 6709.56 0.81 0.24 3391.96 64.63 Msi1, Ngn2,GAD45b #10, −CytoB, 0.12 0.00 0.73 0.03 5.28 0.21 50.84 1.23 4.93 0.2817400.66 822.88 3.58 0.10 1255.76 5.27 Msi1, Ngn2 GAD45b #11, +CytoB,0.10 0.00 0.26 0.01 1.11 0.01 3.69 0.09 0.76 0.00 55588.41 1331.20 0.550.14 2849.96 261.51 Msi1, Ngn2 MBD2 #12, −CytoB, 0.44 0.01 1.47 0.065.49 0.14 47.30 0.11 5.50 0.31 14587.46 789.19 3.90 0.13 1424.04 39.29Msi1, Ngn2 MBD2 #13, +CytoB, 1.11 0.04 1.09 0.06 0.92 0.08 0.68 0.010.82 0.03 63.93 2.81 1.19 0.17 17.43 1.86 GAD45b #14, −CytoB, 0.94 0.012.22 0.00 2.82 0.02 6.49 0.30 4.01 0.05 6.12 0.61 2.34 0.17 1.42 0.10GAD45b #15, +CytoB, 0.83 0.00 0.83 0.05 0.36 0.01 0.16 0.01 0.36 0.003.42 3.74 0.63 0.37 2.18 0.12 MBD2 #16, −CytoB, 0.68 0.02 1.55 0.04 1.570.05 1.47 0.01 2.00 0.00 0.52 0.29 1.45 0.15 0.55 0.04 MBD2 #17, +CytoB,1.10 0.01 1.16 0.03 1.37 0.01 1.12 0.06 0.86 0.06 5.59 1.48 1.07 0.271.70 0.46 Msi1, Ngn2 #18, −CytoB, 0.93 0.04 2.52 0.10 3.48 0.01 9.010.02 4.55 0.18 1.78 1.46 3.83 0.42 0.59 0.01 Msi1, Ngn2 #19, +CytoB,0.20 0.03 0.36 0.01 1.25 0.05 6.68 0.31 0.72 0.02 66592.29 3481.89 2.570.03 4450.08 131.85 Msi1, MBD2 #20, −CytoB, 0.12 0.00 0.64 0.03 4.700.22 77.51 0.11 4.12 0.11 19128.03 1542.00 8.14 0.13 999.22 24.75 Msi1,MBD2 #21, +CytoB, 0.17 0.01 0.28 0.00 1.16 0.04 5.73 0.06 0.62 0.0067945.51 3000.74 2.15 0.04 4736.83 11.92 Ngn2, MBD2 #22, −CytoB, 0.170.00 0.78 0.03 4.32 0.08 68.89 5.26 4.01 0.04 16570.91 92.96 7.04 0.531427.13 13.19 Ngn2, MBD2 #23, +CytoB, 0.71 0.05 0.79 0.06 0.87 0.01 0.630.06 0.67 0.04 2.86 0.70 1.08 0.08 2.08 0.11 Msi1 #24, −CytoB, 0.66 0.041.92 0.17 2.03 0.02 2.77 0.02 2.68 0.02 0.32 0.12 1.85 0.65 0.58 0.04Msi1

Same pattern of gene expression was observed when transfecting the cellswith three transcription factors (Msi1, Ngn2, and Pax6), but theexpression was less pronounced than in cells transfecting with just Msi1and Ngn2. In terms of immunohistochemical analysis after the 12 days ofthe transfection, cells displayed neuronal markers after transfectionwith Msi1 or Ngn2, as indicated by the expression of nestin and MAP2b(FIG. 9). Cells transfected with pCMV-XL-PAx6 did not stain for Nestinand MAP2b.

This study shows that transfecting cells within CDM with only oneneurogenic factor (Msi1 or Ngn2) induces morphological changes andexpression of one or more markers of neural stem cells and neuronalcells. Since the reprogrammed cells expressed a key neurogenic factor, aneuronal precursor marker, and a mature neuronal marker at lowpercentage (10%), this suggests that cells within the CDM weretransformed to NSLCs and then started to differentiated through thevarious phases of the neuronal determination and differentiation programinduced in neural stem cells.

Example XI Gene Expression Analysis of Reprogrammed Cells within CDM

This study was designed to test the effect of transfecting cells withMsi1 and Ngn2 in the presence of MBD2 in the reprogramming process.Cells were transfected after two days of pre-treatment with cytochalasinB with the DNA expression vectors using Lipofectamine reagent asdescribed in Example X. 15 μg of eukaryotic DNA expression vectorspCMV6-XL5-Musashi or pCMV6-XL4-Ngn2, and pCMV6-XL5-MBD2 (Origene), wereused to co-transfect cells. After 24 hours, the media was changed toCDM:Neural Progenitor Maintenance Medium (1:1) supplemented with Noggin(50 ng/ml), EGF (20 ng/ml), and bFGF (20 ng/ml). Medium was changedevery day by increasing the percentage of NPBM and decreasing CDMmedium. Cells were cultured for 6 days at 37° C., 5% CO₂ and 5% O₂.After one week, differentiation was initiated by gradually supplementingthe NPBM Medium with NT-3 (20 ng/ml, Peprotech), all-trans-retinoic acid(ATRA, 5 μM, Spectrum), BDNF (20 ng/ml, Peprotech), and bFGF (40 ng/ml,Peprotech). Samples were collected at the end of the study (day 14) anddata were analyzed by gene array to identify genes that werereproducibly found to be specific for neuronal lineages.

Gene Expression Analysis

Gene expression analysis on 8 samples was performed as previouslydescribed in Example I with the customized Neuronal Markers 2 TLDA Inorder to identify the expression of genes related to neural stem cells,neuronal cells and glial cells, and growth factors expressed by thecells after transfection. The expression of oligodendrocyte genes, suchas NKx2.2, olig2, and MAG was increased by Msi1 and Ngn2; however, theincreased was more pronounced by Msi1 as compared to Ngn2 (Table 26).Two markers for astrocytes (GFAP and AQP4) were highly expressed aftertransfection with Msi1 and Ngn2 in the presence of the DNA demethylatorMBD2. Interestingly, several markers of early neuronal cells wereenhanced; 12 days after transfection, TDLA data revealed increases inspecific markers for interneurons, such as somatostatin and calbindin1.Doublecortin (DCX), which is expressed by migrating immature cellsduring development, and acetylcholine (ACHE), an early marker ofneuronal cells, were highly expressed in reprogrammed cells (Table 26).Transfection with Msi1 or Ngn2 increased the expression ofdihydropyrimidinase-like 3 (DPYSL3), an early marker of newborn neuronsto five-fold with Msi1 and seven-fold with Ngn2. Expression ofmicrotubule-associated protein 2 (MAP2), an essential marker fordevelopment and maintenance of early neuronal morphology, and neuronalcell adhesion molecule (NCAM) were highly expressed with Msi1 and Ngn2.The expression of enolase-2, a marker of mature neurons, was 20-foldenhanced by Msi1 and Ngn2. Member of the NeuroD family NeuroD1 washighly expressed after transfection with Msi1 to 84.22 fold and to 34.27by Ngn2. Gene expression of growth factors such as IGF-1, IGF-2, NPY andCSF-3 was enhanced following transfection with Msi1 or Ngn2. Theexpression of VEGF and GDNF genes were increased to almost five-fold andseven-fold by Msi1 and Ngn2, respectively. However in transfected cells,the expression of BDNF, EGF, and bFGF were not activated and evendown-regulated as compared to untransfected cells. The expression ofgrowth associated protein (GAP-43), a growth- andregeneration-associated marker of neurite extension, and expression ofnetrin, implicated in neuronal development and guidance, were highlyexpressed in transfected cells (Table 26). Expression of receptors forgrowth and neurotrophic factors was increased, such as type III receptortyrosine kinase, Neurotrophic tyrosine kinase receptor, and neurotrophictyrosine kinase. The fibroblast-specific markers vimentin andfibronectin were down-regulated in the reprogrammed cells.

Transfection of HFF with only Msi1 and Ngn2 in the presence of MBD2increased the expression of glial cells and neuronal cells markers.

TABLE 26 Gene array of CDM transfected with pMsi1 and pNgn2 followingthe pre-treatment with cytochalasin B (10 μg/ml), VPA (4 mM) and5-Azacytidine (5 μM). Transfected cells were cultured in differentiationmedium (NbActive, BrainBits ™) supplemented by ATRA (5 μM), bFGF (40ng/ml) and BDNF (20 ng/ml). Relative Relative expression expressionSymbol Common name and description Company Gene ID Msi1 Ngn2 Astrocytesand oligodendrocytes markers NKx2-2 Markers for oligodendrocyteprogenitors NM_002509.2 1.72 10.19 OLIG2 Oligodendrocyte lineagetranscription factor 2 NM_005806.2 1.72 1.52 MBP Myelin-basic proteinNM-001025090.1 1.72 1.52 GFAP Glial fibrillary acidic proteinNM_002055.4 6.04 2.41 AQP4 Aquaporin 4 NM_001650.4 1.72 1.52 DIO2Deiodinase iodothyronine type II NM_013989.3 8.29 10.61 NC markers SSTSomatostatin, specific marker for interneurons NM_001048.3 very highvery high CALB1 Calbindin 1, interneuron marker NM_004929.2 1.72 1.52Tubulin1A Are necessary for axonal growth NM_006009.2 0.63 0.76 NESPrecursor neurons (nestin) NM_006617.1 2.42 2.86 DCX An early neuronalmarker (Doublecortin) NM_178151.1 1.72 1.52 ACHE Acetylcholinesterase,marker of early neuronal NM_015831.2 10.68 20.37 development ENO2 Amarker for neurons cells, enolase NM_001975.2 0.55 0.54 NEUROD1 Neuralmarker; expression gradually increased NM_002500.2 1.72 1.50 from neuralprecursor to fully differentiated neuron DPYSL3 Dihydropyrimidinase-like3, marker of NM_001387.2 0.62 0.71 immature neurons MAP2Microtubule-associated protein 2, essential for NM_002374.3 1.99 1.70development of early neuronal morphology and maintenance of adultneuronal morphology NCAM Neural cell adhesion molecule 1 NM_18135.3 3.115.72 CEND1 Cell cycle exit & neuronal differentiation, early NM_016564.36.68 8.28 marker of proliferating precursor cells that willdifferentiate to neurons Neuroregeneration and survival genes FGF2Fibroblast growth factor NM_002006.4 1.19 1.26 EGF Epidermal growthfactor, Hs00153181_m1 28.37 52.13 IGF-1 Insulin growth factor-1,NM_000618.2 0.82 1.03 IGF-2 Insulin growth factor-2 NM_0000612.3 0.991.21 CSF3 Granulocyte colony-stimulating factor NM_2219.1 very high veryhigh BDNF Brain derived growth factor, neurogenesis NM-199231.1 8.547.84 GDNF Glial derived neurotrophic factor NM-000614.2 0.63 0.91 CNTFCiliary neurotrophic factor NM_001025366.1 3.80 14.92 VEGF Vascularendothelial growth factor NM_130850.1 6.28 7.22 BMP-4 Bone morphogeneticprotein 4 NM_002253.1 1.17 1.34 KDR Type III receptor tyrosine kinase)NM_006180.3 113.85 43.87 NTRK2 Neurotrophic tyrosine kinase recepto(TrkB) NM_000905.2 0.02 0.02 NPY Neuropeptide Y NM_009905.2 33.39 1.52NTF-5 Neurotrophin 5 NM_006179.3 4.43 5.93 PIK3CGphosphoinositide-3-kinase, NM_002649.2 1.70 1.50 STAT3 Signaltransduction transcription 3 NM_213662.1 3.15 2.24 Gap43 Growthassociated protein 43 NM_002045.2 1.82 2.98 NTN1 Netrin1, implicated inneuronal development NM_004822.2 0.50 0.29 and guidance NTRk2Neurotrophic tyrosine kinase, receptor, type 2 NM_006180.3 0.02 0.02L1CAM L1 cell adhesion molecule, associated with NM_024003.1 0.08 0.11regenerating axons LIMK1 LIM domain kinase 1 NM_002314.2 2.88 2.96Vimentin Radial glia and fibroblast marker NM-003380.2 0.21 0.20Fibronectin fibronectin is a marker for fibroblasts NM_212474.1 0.150.14

Example XII Reprogramming of Cells within CDM by Lipofectamine andNucleofection

This study was designed to improve transfection of CDM by combininglipofectamine and nucleofection and using two vectors pCMV6-XL5-Msi1 andpCMV6-XL4-Ngn2 individually or in combination together withpCMV-XL5-MBD2. Cells within Day 4 CDM were lipotransfected for 6 hourswith Msi1/MBD2, Ngn2/MBD2 or Msi/Ngn2/MBD2 after 2 days of pre-treatmentwith or without cytochalasin B. In parallel, transfection was performedon fresh HFFs after the 6 hours using Nucleofection as described inExample II, and transferred on top of the CDM when the lipofectaminemedia was changed to fresh CDM medium. After 24 hours, the medium waschanged to Neural Progenitor Basal Medium (NPBM, Lonza) with thepresence of Noggin (50 ng/ml, Peprotech), recombinant hFGF (20 ng/ml,Peprotech), and recombinant hEGF (20 ng/ml, Peprotech). Differentiationwas induced at day 7, by adding NSA-A differentiation medium (StemCellTechnologies) for 21 days.

Gene Expression Analysis

Samples were collected at 8, 15, and 21 days to evaluate the nature ofnewly formed cells by analyzing the expression of several neuronalmarker genes using RT-PCR according to the methods previously describedin Example I. As shown in Table 27, cells transfected with oneneurogenic transcription factor (Msi1 or Ngn2) express high levels ofnestin and βIII-tubulin at day 8. The same pattern of expression wasobserved at day 15 and 21, while the expression was slightly decreasedin the absence of cytochalasin B in cells transfected with Ngn2. Theexpression of all genes, except the mature neuronal marker MAP2b, wereremarkably increased in cells transfected with both neurogenictranscription factors. The upregulation of these genes was slightlyreduced in the absence of cytochalasin B, indicating its role inenhancing reprogramming.

TABLE 27 RT-PCR analysis of relative expression of neuronal stem cellmarkers such as nestin, Sox2, and GFAP after transfection of fibroblastcells within the CDM with different combinations with or without theco-treatment with cytochalasin B. Relative expression of Sox2, nestin,and GFAP in NSLCs was increased after transfection with bothtranscription factors Ngn2 and Msi1 with MBD2 as the DNA demethylator.MSI1 NGN2 TUBB3 GFAP NES MAP2 Rel. Std. Std. Rel. Std. Rel. Std. Rel.Std. Rel. Std. Exp. Dev. Rel. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.Exp. Dev. #1 Day8 CDM − CytoB Control 1.11 0.21 1.33 0.20 1.10 0.02 0.910.02 1.18 0.09 0.91 0.02 #2 Day8 CDM − CytoB Control 1.11 0.17 0.65 0.080.92 0.06 0.91 0.11 0.82 0.01 0.91 0.11 #3 Day8 CDM − CytoB Control 0.830.01 0.71 0.86 0.99 0.04 1.21 0.00 1.03 0.00 1.21 0.00 #4 Day8 CDM +CytoB Control 7.42 0.35 1.52 0.53 1.32 0.16 0.44 0.06 1.04 0.02 0.440.06 #5 Day8 CDM + CytoB Control 7.01 0.42 2.14 0.58 1.23 0.07 0.62 0.051.02 0.06 0.62 0.05 #6 Day8 CDM + CytoB Control 9.15 0.48 0.76 0.08 0.400.05 0.59 0.14 0.34 0.16 0.59 0.14 #7 Day15 CDM − CytoB Control 1.450.07 1.53 0.33 1.32 0.01 0.90 0.07 1.31 0.03 0.90 0.07 #8 Day15 CDM −CytoB Control 0.79 0.02 2.01 1.49 0.91 0.03 1.14 0.16 0.91 0.01 1.140.16 #9 Day15 CDM − CytoB Control 0.87 0.04 0.64 0.72 0.84 0.08 0.980.15 0.84 0.01 0.98 0.15 #10 Day15 CDM + CytoB 1.27 0.14 0.99 0.66 1.700.21 0.36 0.02 1.08 0.08 0.36 0.02 Control #11 Day15 CDM + CytoB 1.390.04 0.97 0.65 2.65 0.38 0.44 0.06 1.97 0.30 0.44 0.06 Control #12 Day15CDM + CytoB 1.09 0.21 0.49 0.46 1.32 0.14 0.47 0.15 2.45 0.15 0.47 0.15Control #13 Day21 CDM − CytoB 1.21 0.00 1.06 0.06 1.10 0.01 0.86 0.161.07 0.01 0.86 0.16 Control #14 Day21 CDM − CytoB 0.97 0.09 2.16 0.770.96 0.01 1.11 0.10 0.94 0.01 1.11 0.10 Control #15 Day21 CDM − CytoB0.86 0.02 1.01 1.27 0.94 0.00 1.08 0.26 0.99 0.04 1.08 0.26 Control #16Day21 CDM + CytoB 1.41 0.21 1.29 1.64 2.46 0.07 0.88 0.22 1.58 0.05 0.880.22 Control #17 Day21 CDM + CytoB 2.24 0.00 0.35 0.01 2.23 0.03 0.550.16 1.57 0.02 0.55 0.16 Control #18 Day21 CDM + CytoB 2.18 0.14 0.770.06 2.29 0.12 0.54 0.04 1.47 0.04 0.54 0.04 Control #19 Day8 CDM −CytoB 694.16 18.10 0.51 0.05 1.46 0.04 2.18 0.13 1.02 0.03 2.18 0.13Msi1/MBD2 #20 Day8 CDM − CytoB 2.38 0.29 4106.88 48.57 0.46 0.02 1.880.14 0.99 0.02 1.88 0.14 Ngn2/MBD2 #21 Day8 CDM − CytoB 365.04 6.712702.81 55.69 4.44 0.02 2.95 0.38 5.11 0.05 2.95 0.38 Msi1/Ngn2/MBD2 #22Day8 CDM + CytoB 1262.00 63.21 0.75 0.91 0.54 0.03 2.48 0.11 1.16 0.052.48 0.11 Msi1/MBD2 #23 Day8 CDM + CytoB 2.34 0.20 10963.51 19.89 0.530.00 2.27 0.26 1.00 0.06 2.27 0.26 Ngn2/MBD2 #24 Day8 CDM + CytoB 869.1565.33 6401.28 87.12 4.58 0.01 3.65 0.13 3.15 0.00 3.65 0.13Msi1/Ngn2/MBD2 #25 Day15 CDM − CytoB 41.07 1.74 2.58 0.36 1.43 0.05 0.580.06 1.34 0.07 0.58 0.06 Msi1/MBD2 #26 Day15 CDM − CytoB 0.73 0.022192.64 15.74 0.95 0.08 1.01 0.09 0.99 0.03 1.01 0.09 Ngn2/MBD2 #27Day15 CDM − CytoB 45.59 2.33 3318.42 51.51 5.32 0.08 3.80 0.01 4.32 0.014.80 0.01 Msi1/Ngn2/MBD2 #28 Day15 CDM + CytoB 106.34 4.43 4.90 1.701.47 0.01 0.57 0.10 1.19 0.03 0.57 0.10 Msi1/MBD2 #29 Day15 CDM + CytoB1.09 0.11 6715.95 505.86 1.30 0.05 0.70 0.17 1.18 0.07 0.70 0.17Ngn2/MBD2 #30 Day15 CDM + CytoB 46.77 0.76 2816.33 90.83 5.76 0.02 4.520.09 3.60 0.03 5.52 0.09 Msi1/Ngn2/MBD2 #31 Day21 CDM − CytoB 22.94 1.0910.09 2.72 1.08 0.07 0.58 0.08 1.17 0.02 0.58 0.08 Msi1/MBD2 #32 Day21CDM − CytoB 0.78 0.02 4450.56 255.75 1.00 0.03 0.75 0.21 1.09 0.03 0.750.21 Ngn2/MBD2 #33 Day21 CDM − CytoB 24.02 0.86 2509.95 64.00 5.18 0.054.74 0.16 4.37 0.06 3.74 0.16 Msi1/Ngn2/MBD2 #34 Day21 CDM + CytoB 54.171.41 8.31 3.32 1.42 0.05 0.70 0.22 1.71 0.02 0.70 0.22 Msi1/MBD2 #35Day21 CDM + CytoB 1.19 0.15 1180.19 27.29 1.21 0.06 1.03 0.34 1.31 0.041.03 0.34 Ngn2/MBD2 #36 Day21 CDM + CytoB 81.66 1.34 7789.96 345.72 5.240.05 5.84 0.10 4.37 0.05 5.84 0.10 Msi1/Ngn2/MBD2

Immunohistochemical Analysis

Samples were collected at 4, 8, 14, and 21 days to evaluate the natureof any reprogrammed cells by analyzing the expression of severalneuronal markers using immunohistochemical analysis according to themethods previously described in Example I. The immunohistochemicalanalysis at various time points revealed that within the first 8 daysthe expression of nestin was induced in a large proportion of cells anddecreased time-dependently after inducing the differentiation (FIG. 10).

This study indicates that upon transfecting the cells with one or twoneurogenic genes in the presence of cytochalasin B and MBD2,reprogrammed cells were stable in culture, responded to environmentalchanges (proliferation vs differentiation), and expressed neuronalmarkers for at least 24 days in culture.

Example XIII Telomerase Activity of NSLCs

Telomerase is active in neural precursor cells and suggest that itsregulation is an important parameter for cellular proliferation to occurin the mammalian brain (Caporaso G L et, 2003). This study was performedto evaluate telomerase activity in cell extracts of adherent NSLCs(NSLCs cultured on laminin-coated plates) as well as NSLCs in floatingneurospheres (NSLCs cultured in plates with a low-bind surface) at early(P7) and late passage (P27). The telomerase activity of the 4 sampleswas measured by the PCR-based telomere repeat amplification protocol(TRAP) using the TRAPeze® Telomerase Detection Kit (Chemicon). Briefly,the cells were grown in 24-well plates, washed in PBS, and homogenizedfor 30 min on ice in buffer containing 10 mM Tris-HCl, pH 7.5, 1 mMMgCl₂, 1 mM EGTA, 0.1 mM Benzamidine, 5 mM β-mercaptoethanol, 0.5% CHAPSand 10% Glycerol (1×CHAPS Lysis Buffer, provided in kit) and RNaseInhibitor. The samples were spun down and the protein concentration ofthe supernatant was determined using the BCA Assay. 900 ng of proteinfrom each cell extract was added directly to the TRAP reaction mixturecontaining TRAP reaction buffer, dNTPs, template substrate (TS) primer,TRAP primer mix and Taq polymerase. The reaction mixtures were incubatedat 30° C. for 30 minutes for template synthesis, followed by a PCRprocedure (95° C./15 min for initial denaturation, 94° C./30 sec, 59°C./30 sec, 72° C./1 min for 32 cycles) for amplification of the extendedtelomerase products. To detect telomerase activity, polyacrylamide gelelectrophoresis (PAGE) was performed for the reaction products on a 10%non-denaturing TBE gel. After electrophoresis, the gel was stained withSYBR® Green I Nucleic Acid Gel Stain for 30 minutes, followed by imagecapture using a Gel-Documentation System (Alpha Innotech).

All 4 samples were telomerase positive (as indicated by the TRAP productladder) as shown in FIG. 11. As expected, the Heat-treated control (ΔH)showed no Telomerase activity (Negative Control). A 36 bp internalcontrol band (S-IC) is used to monitor PCR amplification (to distinguishfalse-negative results). This S-IC band was observed for all samplesexcept for the test samples. This may have been due to the excessivelyhigh telomerase activity in the test samples; amplification of the TRAPproducts and the S-IC control band are semi-competitive. All controlsgave expected results (No TRAP products for CHAPS ctrl, and TRAP ladderof products for the positive control cells and the TSR8 control).

Example XIV Tumor Formation Assay

Malignantly transformed cells show reduced requirements forextracellular growth promoting factors, are not restricted by cell-cellcontact, and are often immortal. Anchorage-independent growth andproliferation is one of the hallmarks of malignant transformation, whichis considered the most accurate and stringent in vitro assay fordetecting malignant transformation of cells.

Adherent and neurosphere NSLCs at early and late passage (P7 and P25),as well as normal human neuroprogenitor cells (hNPCs), were investigatedfor the anchorage-independent growth. HFFs were used as a negativecontrol and cervical carcinoma HeLa cells were used as a positivecontrol. Cells were sedimented by centrifugation at 150×g for 3 min atroom temperature (RT). The assay was performed using the CytoSelect™96-well cell transformation assay (CellBiolabs). The base agar layer(1.2%) was dissolved in 2×DMEM/20% PBS solution and 50 μl of the agarsolution was added to the plate and incubated for 30 min at 4° C. tosolidify. Prior to adding the cell agar layer, the plate was allowed towarm up for 15 minutes at 37° C. The cells were resuspended at differentdensity (20.000 and 5000 cells/well), except the hNPCs were resuspendedonly at 5000 cells/well due to a lack of enough cells. The cells weremixed with the 1.2% agar solution, 2×DMEM/20% PBS, and cell suspension(1:1:1), and 75 μl of the mixture was transferred to wells alreadycontaining the solidified base agar layer, and was then placed in 4° C.for 15 minutes to allow the cell agar layer to solidify. 100 μl ofproliferation medium (StemCell Technologies) was added and the plate wasincubated for 8 days at 37° C. and 5% CO₂ before being solubilized,lysed and detected by the CyQuant™ GR dye in a fluorescence platereader. The fluorescence measurement was performed using theFlexstation™ (Molecular Devices) with a 485/538 nm filter.

TABLE 28 Fluorescence measurement (Relative Fluorescence Unit, RFU)indicate that under the same conditions only carcinoma HeLa cells growas an anchorage-independent colony, while both hNPCs and NSLCs (adherentand floating neurospheres) were negative for tumor growth in thestandard agar plate tumor formation assay (CytoSelect ™ celltransformation kit, Cell Biolabs Inc.). Cell types Cell density Hela HFFNSLCs HNPCs 20.000 60.05 ± 8.70 14.82 ± 1.57 19.22 ± 1.85 19.00 ± 2.7110.000 39.03 ± 3.97 13.73 ± 1.05 14.99 ± 1.12 21.61 ± 9.95 5000 24.70 ±3.89 11.65 ± 0.57 12.29 ± 0.79 12.45 ± 0.73

As shown in Table 28, fluorescence measurement indicated that under thesame conditions only carcinoma HeLa cells significantly grew andproliferated as anchorage-independent colonies, while both hNPCs andNSLCs (adherent and floating neurospheres) were negative for tumorgrowth (same value as HFFs (negative control) for 5,000 and 10,000cells) in the standard agar plate tumor formation assay by visualobservation of cells by light microscopic observation using bright fieldat 10× confirm Fluorescence measurement. Thus the transient transfectionmethod and genes used allows the reprogramming of cells without theneoplastic transformation that generally occurs with stable transfectionor certain genes via a series of genetic and epigenetic alterations thatyield a cell population that is capable of proliferating independentlyof both external and internal signals that normally restrain growth.

Example XVI No Genomic Integration of Plasmid DNA in NSLCs fromTransient Transfection

The DNA plasmid Msi1/Ngn2 (designed and constructed in house) was usedin transient transfection for generation of NSLCs along with MBD2 (forsample 1), or 5-Aza and VPA (for sample 2). Two weeks aftertransfection, Southern blot was performed to test for possible genomicintegration of the plasmid DNA. 3 μg of genomic DNA extracted from theNSLC samples, as well as from HFF (a human fibroblast cell line) used asa negative control, was digested with several restriction enzymesincluding BgIII, PstI and StuI, subjected to electrophoresis on a 1%agarose gel and transferred to a positively charged nylon membrane(Roche). The membrane was hybridized in the DIG Easy Hyb™ buffer (Roche)at 42° C. overnight with a 1.2 kb Dig-labeled PCR probe amplified fromthe plasmid DNA using a set of primers. The membrane was washed twice atroom temperature with 2×SSC, 0.1% SDS for 5 min per wash, twice with0.5×SSC, 0.1% SDS at 65° C. for 15 min per wash. Hybridization signalsof the membrane were detected using the CDP-Star™ substrate (Roche). Themembrane was exposed to an X-ray film for analysis. The signals werestripped from the membrane using stripping buffer (0.2 M NaOH, 0.1%SDS). The membrane was re-hybridized with a 0.9 kb Dig-labeled PCR probeamplified from the plasmid DNA using a set of primers.

The Southern blot analysis (FIG. 12) with the 1.2 kb Dig-labeled PCRprobe revealed distinct signals in the positive control samples wherethe Msi1/Ngn2 plasmid DNA was spiked into HFF genomic DNA for theequivalence of 1, 10 or 100 integrations per genome. There were a fewweak and identical bands that appeared in the restriction enzymedigested genomic DNA from HFF, NSLC samples #1 and #2, suggesting thatthere is no plasmid DNA integration in the genomic DNA of NSLCs. Thesebands may represent the endogenous Ngn2 gene since the 1.2 kbDig-labeled PCR probe contains a small part of the Ngn2 gene. This datashows that no, or only a tiny number of, NSLCs had plasmid integrationinto the host genome after transient transfection, and that thetransfected genes are only present in the cell for a short period oftime (less than two weeks).

Example XVII Neuroprotective Effect of Transplanted hNSLCs in 1) AnimalModel of Multiple Sclerosis.

Multiple Sclerosis (MS) is an incurable inflammatory demyelinatingdisease of the central nervous system (CNS) (Frohman E M et al 2006).Therapies for MS rely on manipulation of the immune system, but withoften modest effectiveness on reducing clinical episodes or permanentneurological disability, requiring frequent injections, and withsometimes-significant side effects (Langer-Gould A et al 2004).Experimental Allergic Encephalomyelitis (EAE) is an animal model of MScommonly used for studying disease mechanisms and testing potentialtherapies. EAE can be induced in a variety of species and strains ofanimals [mice, Rat, marmoset monkey, rhesus macaques] using various CNSantigens [Myelin Oligodendrocyte Glycoprotein (MOG), proteolipid protein(PLP) and myelin basic protein (MBP)].

After obtaining all appropriate animal approvals for the experiments,Female 7 to 8 weeks old C57BL/6 mice were purchased from Charles Rivers,and housed at MISPRO animal facility for one week before experimentationfor adaption to the new environment. C57BL/6 mice were injected s.c.with 100 μg MOG 35-55 in CFA (Sheldon Biotechnology, McGill University)containing 5 mg/ml Mycobacterium tuberculosis H37Ra (Difco, Inc), at 2sites on the back. All mice received 200 ng pertussis toxin (ListBiological Laboratories, Inc) i.p. on day 0 and 2, while clinical scoreswere calculated blindly daily during a 43 day period, according to the0-5 scale as follows: 1, limp tail or waddling gait with tail tonicity;2, waddling gait with limp tail (ataxia); 2.5, ataxia with partial limbparalysis; 3, full paralysis of 1 limb; 3.5, full paralysis of 1 limbwith partial paralysis of second limb; 4, full paralysis of 2 limbs;4.5, moribund; and 5, death.

Treatment of EAE Animal Model with and without the Cells:

hNSLC and hNPCs (1.5×10⁶ cells in 200 μl PBS/each mouse) were given bysingle injection i.v. via the tail vein when the animals started to showsymptoms of EAE (day 13 i.v). Both animals groups received cyclosporine(10 mg/kg/day) one day before the injection of cells and daily from theday of transplantation to avoid any rejection of the human cells.Sham-treated age-, sex-, and strain-matched mice, injected i.p. with PBSalone, were used as controls. All groups of animals were observed for 43days. Animals were sacrificed at 43 days p.t., brains and spinal cordwere harvested in 30% sucrose in PBS. Statistical analysis of theclinical scores revealed that the clinical signs of EAE weresignificantly attenuated in NSLC-injected animals as compared to controland hNPCs-injected animals. Cumulative scores was significantly reducedin the NSLC transplanted animals (FIG. 13) and the treatment has noeffect on body weight.

2) Hemiplegic Animal Model (Unilateral Ablation of the Left SensorimotorCortex in Adult Rats).

After obtaining all appropriate animal approvals for the experiments, 8rats per group (Sprague-Dawley, 250-300 g, Charles River) wereanaesthetized using ketamine (Bimeda-MTC)/xylazine (50/10 mg/kg,Novopharm) and placed onto a stereotaxic frame. A midline cranialincision was performed with a sterile surgical scalpel blade, thecranial vault exposed and the bregma identified. The skull above thesensorimotor cortex was opened and the sensorimotor cortex area [0.5-4.0mm caudal to bregma and 1.8-3.8 mm lateral to the midline (Paxinos andWatson 1986)] was carefully aspirated. After ablation, the treatments(Alginate, Alginate+hNPC, Alginate+NSLCs, RM_(x)+NSLCs, RM_(x) Only,Fibrin Gel, or Saline) were applied directly on the brain afterablation. The opening in the skull was then filled with Bone Wax. Incase of a bleeding, small pieces of sterile homeostatic tissue wereinserted into the lesion in order to stop the bleeding. The sutures wereperformed using Ethicon™ monofilament suture ½ circle needle shape.Surgeries were performed in sterile clean rooms, and topical antibiotics(Cicatrin®, GlaxoSmithKline) were applied to the exposed skull and scalpto limit local infection. Rats were immunosuppressed by daily injectioni.p. of cyclosporine A (10 mg/kg/day) starting the day before thesurgery until the end of the study period. The purpose of thecyclosporine A injection was to reduce the rat's immune reaction to thetreatment. The immune-suppression was sustained until the end of thestudy to ensure that any potential failure of regeneration (if takenplace) was not due to the immune reaction against the treatment.Functional scores were performed weekly, in all groups, sensorimotorimpairment was evaluated based on the behavioural tests as describedbelow.

Rotarod Test:

The rotarod speed was manually calibrated for the 10 and 20 RPM speedbefore all procedures. Animals were required to perch on the stationaryrod for 30 sec to acclimate themselves to the environment. During thistime, if any animal fell, it was placed back on the rod until it hadachieved stationary capabilities for a period of 30 seconds. The animalswere allowed 3 trials. The animals that were comfortable staying on thestationary rod for 30 sec were allowed to run with a constant speed of10 and 20 RPM for 60 sec, and the number of falls were electronicallyrecorded.

Beam Walking:

Beam walking measures hindlimb coordination by means of distancetravelled across 100 cm beam (2.3 cm in diameter, 48 cm off the floor).Rats were systematically trained to walk along the elevated beam fromstart to finish with the aim of completing the task. A safe location,i.e, a flat box, is placed at the end of the beam so that the rat ismotivated to complete the task.

Scale used for evaluation of beam-walking performance Scale Performancecharacteristic 1 Animals fail to traverse the beam and do not place thehindlimb on the horizontal surface of the beam 2 Animals fail totraverse the beam, but place the hindlimb on the horizontal surface ofthe beam and maintain balance 3 Animals traverse the beam while draggingthe hindlimb 4 Animals traverse the beam and place the hindlimb at leastonce during the traverse 5 Animals traverse the beam using the hindlimbto aid less than 50% of its steps on the beam 6 Animals traverse thebeam using the left hindlimb to aid more than 50% of its steps on thebeam 7 Animals traverse the beam with no more than two foot slips 8Normal animals

Before the surgery, all the animals fell at least once from the rotarod,not because they had a walking or coordination problem, but because thespeed was high. After the surgery (2 days), all the animals showed signsof significant walking and coordination problems leading to an increasein the number of falls from the rotarod. Three weeks after the surgery,the number of falls was clearly reduced for the animals receiving NSLCsas treatment compared to controls (FIG. 14).

Animals passed the beam-walking test before the surgery without anydifficulty. The rats crossed the 100 cm beam and got to the safe spotwithout falling off the beam. Two days after surgery, all groupscompletely failed to pass the test, and the animals were not able tostay in balance on the beam. One week after the surgery, all the animalsshowed some improvement in their walking capacity, but no significantdifference was noticeable between the different treated groups. Fromweek 4 until week 26, the animals treated with NSLCs as well as RM_(x)showed significant improvements in their walking capacity compared tothe controls (FIG. 15).

Example XVIII Transfection of HFF by Various Combinations of Genes Usingthe Shuttle® Device and Treatment with Different Small Molecules forReprogramming to Mesendoderm-Like Cells

HFF cells were cultured as described in CDM II medium as described inExample I with only modifying EGF (5 ng/ml) and FGF (10 ng/ml), andtransfecting using the Nucleofector™® 96-well Shuttle® Device (Lonza)following the procedure described in Example IV. The cells weretransfected with various combinations of cDNA clones as described inTable 29. After transfection, the cells were plated on 0.1%Gelatin-coated plates and incubated at 37° C., 5% CO₂, 5% O₂. Medium waschanged every other day according to Table 30. Cells were analyzed atDay 4 by Quantitative Real-time PCR.

TABLE 29 Various combinations of plasmids with potential to transfectthe cells towards mesendoderm lineage. Day −2 to Day 0 Plasmidstransfected at Day 0¹ 1 Untreated Oct4, FoxD3, MBD2 2 Oct4, T, MBD2 3Oct4, Mixl1, MBD2 4 Oct4, Sox17, MBD2 5 FoxD3, T, MBD2 6 FoxD3, Mixl1,MBD2 7 FoxD3, Sox17, MBD2 8 T, Mixl1, MBD2 9 T, Sox17, MBD2 10 Mixl1,Sox17, MBD2 13 Pre-treated with Oct4, FoxD3 14 VPA & 5-Aza FoxD3, T 15FoxD3, Mixl1 16 FoxD3, Sox17 17 Oct4, FoxD3, T 18 Mixl1, Sox17, FoxA2 19Oct4, FoxD3, T 20 Mixl1, Sox17, FoxA2 ¹where Oct4 = pCMV6-XL4-Oct4,FoxD3 = pCMV6-XL5-FoxD3, MBD2 = pCMV6-AC-MBD2, T = pCMV6-XL5-T, Mixl1 =pCMV6-XL5-MIXL1, Sox17 = pCMV6-XL4-SOX17, FoxA2 = pCMV6-XL5-FOXA2. Allclones were purchased from Origene and prepared using the EndoFreePlasmid Maxi Kit (Qiagen).

TABLE 30 Medium composition from Day −2 to Day 10 Media Composition² Day0 Day 1 Day 2 to Day 3 Day 4 to Day 7 Day 8 to Day 10 CDM II (3:1 of CDMII (50%) + IMDM/F12 + IMDM/F12 + IMDM/F12 + NEAA + DMEM:F12; IMDM/F12NEAA + ITS + NEAA + ITS + ITS + HSA + GlutaMAX ™ 100x, (50%) + NEAA +HSA + bFGF + HSA + bFGF + bFGF + EGF + Dexthamesone, ITS + HSA + EGF +VPA + EGF + Activin BMP4 19.7 μg/ml, bFGF + EGF + Activin A + A +CHIR99021 + Glutathione (500 μg/ml, VPA + Activin CHIR99021 + BMP4L-Ascorbic A + BMP4 75 mg/ml, Selenious CHIR99021 acid 2.5 μg/ml,Insulin solution 10 mg/ml, T3 675 ng/ml, ethanolamine 500X, bFGF 2.5ug/ml, and Egf (1.25 ug/ml) + Activin A + HSA ²Supplements added tomedia at the following concentrations: Activin A (Peprotech, 30 ng/ml),HSA (Baxter, 0.5%), NEAA (Gibco, 1X), ITS (Gibco, 1X), EGF (Peprotech, 5ng/ml), bFGF (Peprotech, 10 ng/ml), CHIR99021 (Stemgent, 2uM), VPA(Stemgent, 1 mM), 5-Aza (Sigma, 0.5uM), BMP4 (Peprotech, 10 ng/ml)

Cells were collected on Day 4 by detaching with TrypLE™, followed bycentrifugation at 80×g for 5 minutes. Supernatant was aspirated and thecell pellet was frozen at −86° C. until ready for RNA Isolation. RNAisolation and quantification was performed as previously described inExample I. cDNA was prepared and quantitative real-time PCR wasperformed as previously described in Example II, except the followingTaqman™® Gene Expression Assays (Applied Biosystems) were used:

Gene Taqman ™® Assay ID GAPDH (housekeeper) Hs99999905_m1 PPIA(housekeeper) Hs99999904_m1 FOXA2 Hs00232764_m1 SOX17 Hs00751752_s1Endogenous T Hs00610073_g1 GSC Hs00418279_m1 CXCR4 Hs00607978_s1 GATA4Hs00171403_m1 CER1 Hs00193796_m1 CDH1 (E-cadherin) Hs01023894_m1 p63Hs00978340_m1 SOX2 SOX2_1078-ANY

TABLE 31 Relative Expression FoxA2, Sox17, and Cxcr4 after transfectingHFFs once with various gene combinations with potential to reprogramcells into mesoendoderm-like cells. The exact values are notsignificantly accurate due to low RNA yield, however a trend ofincreasing gene expression was detected for FoxA2, Sox17, and CXCR4.FOXA2 CXCR4 Std. SOX17 Rel. Std. Rel. Exp. Dev. Rel. Exp. Std. Dev. Exp.Dev. Untreated HFF 1.00 0.04 1.00 0.04 1.00 0.04 Day4 HFF untransf. 1.010.06 1.01 0.06 4.77 2.51 (+G.F), Day4 HFF untransf. (−G.F), 1.38 0.111.38 0.11 1.38 0.11 Day4 HFF Untransf. 0.98 0.02 0.98 0.02 3.32 3.31(+G.F.), Day4 HFF Untransf. (−G.F.), 4.12 4.07 1.28 0.06 1.28 0.06 Day4Oct4/FoxD3/MBD2 4.67 4.60 3.19 2.78 76.43 7.91 Day4 Oct4/T/MBD2 3.913.55 4.33 2.36 15.18 2.52 Day4 Oct4/Mixl1/MBD2 2.66 1.77 10.33 0.43 7.313.21 Day4 Oct4/Sox17/MBD2 14.19 4.85 413533.31 127089.61 56.04 0.71 Day4FoxD3/T/MBD2 38.62 38.00 3.12 1.32 42.41 5.23 Day4 FoxD3/Mixl1/MBD2 7.765.29 2.41 0.30 137.17 27.74 Day4 FoxD3/Sox17/MBD2 26.02 1.95 50904.451523.33 131.03 17.53 Day4 T/Mixl1/MBD2 3.67 3.26 5.64 4.15 14.04 2.89Day4 T/Sox17/MBD2 9.76 9.70 209797.21 24533.81 111.35 16.40 Day4Mixl1/Sox17/MBD2 3.60 3.10 237310.10 57448.60 36.76 1.07 Day4 Oct4/FoxD313.87 0.16 13.87 0.16 35.44 14.57 Day4 FoxD3/T 60.93 60.18 19.45 1.5119.45 1.51 Day4 FoxD3/Mixl1 21.20 2.31 28.96 8.66 62.31 55.82 Day4FoxD3/Sox17 96.88 3.60 54177.20 3313.15 44.57 41.51 Day4 Oct4/FoxD3/T25.99 18.15 12.27 1.26 21.17 11.33 Day4 Mixl1/Sox17/FoxA2 1850864.6898259.84 112641.65 15923.21 23.18 23.10 Day4 Oct4/FoxD3/T 9.52 5.61 1.520.02 35.74 4.36 (IMDM/F12) Day4 Mixl1/Sox17/FoxA2 486705.82 19101.5357060.09 1262.81 13.44 2.36 (IMDM/F12)

TABLE 32 Expression of GATA4, CDH1 (E-cadherin), p63, and SOX2 relativeto untreated HFF control 4 days after transfecting HFF cells withvarious gene combinations with potential to reprogram cells intomesoendoderm-like cells. CDH1 (E- GATA4 cadherin) p63 SOX2 Rel. Std.Rel. Std. Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.Untreated HFF 1.00 0.04 1.00 0.04 1.00 0.04 1.00 0.04 Day4 HFF untransf.12.13 0.70 1.01 0.06 3.09 1.45 1.11 0.21 (+G.F), Day4 HFF untransf.(−G.F), 4.48 0.85 1.38 0.11 3.11 2.54 1.38 0.11 Day4 HFF Untransf. 2.372.00 0.98 0.02 4.41 4.40 1.94 1.34 (+G.F.), Day4 HFF Untransf. (−G.F.),6.12 3.33 1.28 0.06 13.23 7.43 1.28 0.06 Day4 95.23 27.44 98.90 21.581.81 0.86 12.72 1.53 Oct4/FoxD3/MBD2 Day4 Oct4/T/MBD2 33.66 10.30 1.420.02 2.05 0.87 2.62 1.67 Day4 Oct4/Mixl1/MBD2 106.33 5.70 1.43 0.03 8.680.99 25.68 2.18 Day4 23.50 5.39 4.65 4.43 95.23 13.86 18.77 6.94Oct4/Sox17/MBD2 Day4 FoxD3/T/MBD2 121.36 11.68 26.85 0.02 2.22 0.0416.99 4.74 Day4 130.21 21.04 69.19 22.84 4.05 3.56 1.52 0.01FoxD3/Mixl1/MBD2 Day4 99.49 30.30 6.89 3.69 1.78 0.01 15.19 9.08FoxD3/Sox17/MBD2 Day4 T/Mixl1/MBD2 110.30 3.55 1.36 0.00 1.36 0.00 6.642.25 Day4 T/Sox17/MBD2 53.19 4.02 2.69 1.86 18.01 0.54 14.21 5.21 Day416.53 16.50 2.91 2.13 13.44 6.68 10.55 3.27 Mixl1/Sox17/MBD2 Day4Oct4/FoxD3 66.45 26.34 47.31 47.30 13.87 0.16 23.87 14.31 Day4 FoxD3/T68.25 68.00 39.08 29.27 19.45 1.51 19.45 1.51 Day4 FoxD3/Mixl1 78.1878.00 21.20 2.31 21.20 2.31 25.10 3.20 Day4 FoxD3/Sox17 176.45 93.5415.64 0.60 15.64 0.60 26.78 16.35 Day4 Oct4/FoxD3/T 12.27 1.26 12.271.26 12.27 1.26 12.27 1.26 Day4 85.89 64.52 20.06 20.00 3.67 0.13 13.660.66 Mixl1/Sox17/FoxA2 Day4 Oct4/FoxD3/T 89.05 50.00 10.40 8.14 1.520.02 1.52 0.02 Day4 6.16 6.10 1.23 0.04 1.23 0.04 1.23 0.04Mixl1/Sox17/FoxA2

Identification of gene combinations that may induce the formation ofMesendoderm-like cells was investigated by transfection withcombinations of Oct4, Sox17, FoxD3, T, MixI1, FoxA2, and MBD2. As shownin Table 25 and 26, the Relative Expression of CXCR4 and GATA4, bothMesendoderm/Endoderm/Mesoderm markers, appear to be up-regulated invarious combinations, most noticeably in FoxD3/MixI1/MBD2 andFoxD3/Sox17/MBD2. Similarly, FOXA2, a marker for Endoderm and Mesoderm,was up-regulated FoxD3/Sox17-transfected sample, although the expressionis still very low. Four days following transfection, SOX17 is stillhighly expressed in the SOX17-transfected samples (50,000 to400,000-fold as compared to the untreated HFF sample). The SOX17 geneexpression represents leftover plasmid DNA (exogenous SOX17) that stillremains 4 days post-transfection, and any endogenous SOX17 expressionthat may have been induced. Ectoderm markers CDH1, p63 and Sox2 werealso up-regulated in some samples (e.g. Oct4/FoxD3/MBD2,Oct4/Sox17/MBD2).

Reprogramming HFFs into Pancreatic Progenitor-Like Cells:

HFF cells were cultured as described in Example I, and transfected usingthe Nucleofector™® 96-well Shuttle® Device (Lonza) following theprocedure described in Example IV. The cells were transfected withvarious combinations of cDNA clones as described in Table 27. Aftertransfection, the cells were plated on Fibronectin-coated collagen gelsand incubated at 37° C., 5% CO₂, 5% O₂. Fibronectin-coated Collagen gelplates were prepared prior to transfection. Rat Tail Collagen I (Gibco)was diluted to 1.125 mg/ml using 10×PBS and distilled water, where 125μl was added to each well of a 24-well plate and incubated in 37° C. for40 minutes. After rinsing with 1×PBS, Fibronectin (BD Biosciences) wasadded on top of the gel at a concentration of 1.9 ug/well. Media waschanged every other day according to Table 33. Cells were analyzed atDay 7 by Quantitative Real-time PCR.

TABLE 33 Plasmids and media composition from Day 0 to Day 14 Plasmidstransfected Media Composition² at Day 0¹ Day 0 Day 1 to Day 3 Day 4 toDay 14 1 FoxD3, Sox17, Pdx1, CDM II + DMEM/F12 + DMEM/F12 + NEAA + ITS +MBD2 Activin A + HSA NEAA + ITS + HSA + B27 + EGF + bFGF + 2 FoxD3,Sox17, Ngn3, HSA + B27 + EGF + Retinoic Acid + FGF10 + MBD2 bFGF +Activin A + Cyclopamine + Noggin 3 FoxD3, Mixl1, Pdx1, CHIR99021 + NaMBD2 Butyrate 4 FoxD3, Mixl1, Ngn3, MBD2 5 Sox17, Mixl1, Pdx1, MBD2 6Sox17, Mixl1, Ngn3, MBD2 7 FoxD3, Sox17, Mixl1, DMEM/F12 + Pdx1 NEAA +ITS + 8 FoxD3, Sox17, Mixl1, HSA + B27 + EGF + Ngn3 bFGF + Activin A + 9FoxD3, Sox17, Pdx1, CHIR99021 + Na Ngn3 Butyrate + VPA + 10 FoxD3,Mixl1, Pdx1, 5-Aza Ngn3 11 Sox17, Mixl1, Pdx1, Ngn3 ¹where FoxD3 =pCMV6-XL5-FoxD3, Sox17 = pCMV6-XL4-SOX17, Mixl1 = pCMV6-XL5-MIXL1, Pdx1= pCMV6-XL5-Pdx1, and Ngn3 = pCMV6-XL5-Ngn3. All clones were purchasedfrom Origene and prepared using the EndoFree Plasmid Maxi Kit (Qiagen).²Supplements added to media at the following concentrations: Activin A(Peprotech, 30 ng/ml), HSA (Baxter, 0.5%), NEAA (Gibco, 1X), ITS (Gibco,1X), B27 (Gibco, 1%), EGF (Peprotech, 5 ng/ml), bFGF (Peprotech, 10ng/ml), CHIR99021 (Stemgent, 2 uM), Na Butyrate (Stemgent, 1 mM), VPA(Stemgent, 1 mM), 5-Aza (Sigma, 0.5 uM), Retinoic Acid (Sigma, 2 uM),FGF10 (Peprotech, 50 ng/ml), Cyclopamine (Stemgent, 2.5 uM), Noggin(Peprotech, 50 ng/ml)

Cells were collected on Day 7 and RNA isolation and quantification wasperformed as previously described in Example I. cDNA was prepared andquantitative real-time PCR was performed as previously described inExample II, except the following Taqman™® Gene Expression Assays(Applied Biosystems) were used:

Gene Taqman ™® Assay ID GAPDH (housekeeper) Hs99999905_m1 PPIA(housekeeper) Hs99999904_m1 FOXA2 Hs00232764_m1 SOX17 Hs00751752_s1GATA4 Hs00171403_m1 Endo PDX1 PDX1_1201 SOX9 Hs00165814_m1 NGN3Hs01875204_s1 NKX2-2 Hs00159616_m1 PAX4 Hs00173014_m1 INS Hs02741908_m1CXCR4 Hs00607978_s1

Identification of gene combinations that may induce the formation ofPancreatic Progenitor-like cells was investigated by transfection withcombinations of FoxD3, Sox17, Pdx1, Ngn3, MixI1, and MBD2. FoxA2, amarker for Endoderm and Mesoderm, was slightly up-regulated for theFoxD3/Sox17/Ngn3/MBD2-transfected sample as compared to the GFPmock-transfected control sample. Similarly, CXCR4, also a marker forboth endoderm and mesoderm, was slightly up-regulated (3-fold comparedto GFP-ctrl) for the FoxD3/MixI1/Ngn3/MBD2-transfected sample. 7 daysfollowing transfection, SOX17 can still be detected for the samplestransfected with SOX17 at varying levels (4 to 570-fold up-regulation ascompared to the GFP-ctrl). The highest SOX17 expression up-regulation isdetected for the sample transfected with Sox17/MixI1/Pdx1/Ngn3 (570-foldas compared to GFP-ctrl), which may suggest that this gene combinationmay increase the amount of SOX17 RNA in cells.

Example XIX Reprogramming Human Adipocytes Derived Stem Cells (ADSC) toPluripotent-Like Stem Cells (PLSC)

ADSCs (Invitrogen Corporation) were cultured in cell culture flasks withcomplete StemPro™-43 medium (Invitrogen) at 37° C., 5% CO₂ and themedium was changed 3 times per week. After 3 days in culture cells(passage 5) were trypsinized and counted to be transfected. Cells weretransiently transfected with one plasmid: pCMV6-Oct4-2A-Klf4-2A-Nanog,pCMV-Sall4-2A-Oct4-2A-Klf4-2A-Nanog, pCMV-Dax1-2A-Oct4-2A-klf4,pCMV-FoxD3-2A-Oct4-2A-klf4, pCMV-Oct4-2A-Klf4-2A-Sall4,pCMV-MBD2-2A-Oct4-2A-Klf4-2A, pCMV-AGR2-2A-Oct4-2A-Klf4-2A, orRex1-EF-Oct4-2A-Klf4 (2 μg); or by two plasmids: pEF-Oct4nuc-IRES2-MBD2with pCMV-Sox2nuc-IREC-Lin28 or pCMV-Klf4nuc-IRES2-Tpt1nuc orpEF-Stella-IRES2-NPM2, using Nucleofector™ as described in Example II.Following the transfection cells were cultured in 6-well plates insuspension with 50:50 ratio of adipocytes complete medium (StemPro™-43)and embryonic stem cells medium (mTeSR1). After two days in culture,cells were re-transfected with the same plasmids listed above and cellswere plated in 96 well-plates coated with Matrigel™ (BD Biosciences) inthe presence of mTesR complete medium supplemeneted with thiazovivin(0.5 μM), an ALK-5 inhibitor (SB 341542, Stemgent, 2 μM), and inhibitorof MEK (PD0325901, Stemgent, 0.5 μM). Medium was changed every day andcells were cultured for 22 days at 37° C., 5% CO₂, 5% O₂. AlkalinePhosphatase Detection Kit (AP, Millipore) and immunohistochemistry wereperformed to analyse the expression of pluripotency markers. ALPstaining was performed using AP detection kit (Millipore) according tomanufacturer's instructions.

Visual observation of reprogrammed cells was performed by Cellomics™using a live staining for SSEA-4₆₄₇ (BD Biosciences) and IRA-1-81₅₅₅ (BDBiosciences) starting on Day 6 after transfection and every 5 daysthereafter. Reprogrammed colonies of PLSCs, positively stained withSSEA-4 and TRA1-81, was observed only with PlasmidpCMV-Sall4-2A-Oct4-2A-Klf4-2A-Nanog, pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP,pEF-Oct4nuc-IRES1-MBD2 with pCMV-Sox2nuc-IRES1-Lin28, andpEF-Oct4nuc-IRES1-MBD2 with pCMV-Klf4nuc-IRES2-Tpt1 nuc. These coloniesemerged around Day 6 and maintained in culture up to the end of thestudy period (Day 22) with a stable morphology. Among the plasmids citedabove, pCMV-Sall4-2A-Oct4-2A-Klf4-Nanog andpEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP gave the highest number of colonies.Live staining showed that these colonies express typical pluripotencymarkers, including SSEA-4 and TRA1-81, and further analysis of thesecolonies showed that the colonies also expressed other ESC markers suchas alkaline phosphatase and Oct4 (FIG. 16). When the cultures weretreated with PD0325901 and SB431542 for up to 22 days, a 4-foldimprovement in efficiency over the conventional method was obtainedfollowing the transfection of ADSCs withpCMV-Sall4-2A-Oct4-2A-Klf4-Nanog and pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP.

Based on the previous study, the highest reprogramming efficiency wasobserved using pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP andpCMV-Sall4-2A-Oct4-2A-Klf4-2A-Nanog. Another study was designed toascertain the effect of pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP on thereprogramming efficiency and to investigate the effect of individualpluripotent genes Rex1, Oct4, and Klf4 in different combinations. ADSCswere transfected as above with pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP,pCMV6-XL5-Rex1, pCMV6-XL4-Oct4/pCMV6-XL5-Klf4,pCMV6-XL5-Rex1/pCMV6-XL4-Oct4, or pCMV6-XL5-Rex1/pCMV6-XL5-Klf4. Afterthe second transfection, ADSC were cultured in 96-well plates coatedwith Matrigel™ for 24 days in the presence of mTeSR1 medium supplementedwith SB341542 and PD 0.325901 at 37° C., 5% CO₂, 5%02. In order tocharacterize subpopulations of cells after transfection, live staining,immunohistochemistry, and AP staining was used to follow the change inpluripotent markers. 1-5% of total cells transfected with Rex1/Oct4 orRex1/Klf4 showed a SSEA4⁺ and TRA-1-81⁺ phenotype, and this pattern wasstable until the end of the study period (Day 22). The observation overtime showed that the phenotype of these colonies moved from an earlySSEA-4⁺ phenotype to a late Oct4⁺/Sox2/Nanog⁺ phenotype by Day 22, whichis closer to the final reprogrammed state of a pluripotent-like stemcell (FIG. 17).

Various genes were tested for their effect on reprogramming efficiencytowards pluripotent-like cells. ADSC cells were cultured as described inExample IX with 2 days VPA and 5-AZA pre-treatment (1 mM and 0.5 μMrespectively) in StemPro™ MSC SFM medium. Cells were transfected usingthe Nucleofector™® 96-well Shuttle® Device (Lonza) following proceduredescribed in Example IV and using the transfection program EW-104 withthe DNA mixes described in Table 34. Following transfection the cellswere plated in StemPro™ MSC SFM medium described in example A onMatrigel™ (BD Biosciences) coated 24 well plates and incubated at 37°C., 5% CO₂, 5% O₂. On Day 1, media was changed to a mix of 75% StemPro™MSC and 25% hES cell medium; the percentage of StemPro™ MSC wasdecreased every day over four days to have 100% hES cell medium by Day4. From then onwards the medium was changed every two days. The hES cellmedium consisted in Dulbecco's Modified Eagle's Medium (DMEM,Invitrogen) supplemented with 20% Knockout™ Serum Replacement (KSR,Invitrogen), 1 mM GlutaMAX™, 100 μM Non-essential Amino acids, 100 μMβ-mercaptoethanol and 10 ng/ml Fgf-2. Different inhibitors and growthfactors were added through the course of the experiment; these arelisted in Table 34. Cells were analysed at Day 7 and Day 14 byimmunohistochemistry analysis and at Day 7 by RT-PCR.

TABLE 34 Plasmids and media composition from Day 1 to Day 14. From dayPlasmids From −2 transfected at From day 1 to From day 3 to day 7 to today 0 day 0 day 3 day 7 day 14 1 VPA + 5- pCMV6-XL4- StemPro ™/hESStemPro ™/hES hES medium Aza pre- Oct4 + medium + ActivinA medium +ActivinA treated pCMV6-XL5- (30 ng/ml) + (30 ng/ml) + Sox2 + CHIR99021(3 μM) CHIR99021 (3 μM) pCMV6-XL5- MBD2 2 VPA + 5- pCMV6-XL4-StemPro ™/hES StemPro ™/hES hES Aza pre- Oct4 + medium + ActivinAmedium + ActivinA medium treated pCMV6-XL5- (30 ng/ml) + (30 ng/ml) +FoxD3 + CHIR99021 (3 μM) CHIR99021 (3 μM) pCMV6-XL5- MBD2 3 VPA + 5-pCMV6-XL4- StemPro ™/hES StemPro ™/hES hES medium Aza pre- Oct4 +medium + ActivinA medium + ActivinA treated pCMV6-XL5-UTF1 + (30ng/ml) + (30 ng/ml) + pCMV6-XL5- CHIR99021 (3 μM) CHIR99021 (3 μM) MBD24 VPA + 5- pCMV6-XL4- StemPro ™/hES StemPro ™/hES hES medium Aza pre-Oct4 + medium + ActivinA medium + ActivinA treated pCMV6-XL4- (30ng/ml) + (30 ng/ml) + DPPA4 + CHIR99021 (3 μM) CHIR99021 (3 μM)pCMV6-XL5- MBD2 5 VPA + 5- pCMV6-XL5- StemPro ™/hES StemPro ™/hES hESmedium Aza pre- Sox2 + medium + ActivinA medium + ActivinA treatedpCMV6-XL5- (30 ng/ml) + (30 ng/ml) + FoxD3 + CHIR99021 (3 μM) CHIR99021(3 μM) pCMV6-XL5- MBD2 6 VPA + 5- pCMV6-XL5- StemPro ™/hES StemPro ™/hEShES medium Aza pre- Sox2 + medium + ActivinA medium + ActivinA treatedpCMV6-XL5- (30 ng/ml) + (30 ng/ml) + UTF1 + CHIR99021 (3 μM) CHIR99021(3 μM) pCMV6-XL5- MBD2 7 VPA + 5- pCMV6-XL5- StemPro ™/hES StemPro ™/hEShES medium Aza pre- Sox2 + medium + ActivinA medium + ActivinA treatedpCMV6-XL4- (30 ng/ml) + (30 ng/ml) + DPPA4 + CHIR99021 (3 μM) CHIR99021(3 μM) pCMV6-XL5- MBD2 8 VPA + 5- pCMV6-XL5- StemPro ™/hES StemPro ™/hEShES medium Aza pre- FoxD3 + medium + ActivinA medium + ActivinA treatedpCMV6-XL5- (30 ng/ml) + (30 ng/ml) + UTF1 + CHIR99021 (3 μM) CHIR99021(3 μM) pCMV6-XL5- MBD2 9 VPA + 5- pCMV6-XL5- StemPro ™/hES StemPro ™/hEShES medium Aza pre- FoxD3 + medium + ActivinA medium + ActivinA treatedpCMV6-XL4- (30 ng/ml) + (30 ng/ml) + DPPA4 + CHIR99021 (3 μM) CHIR99021(3 μM) pCMV6-XL5- MBD2 10 VPA + 5- pCMV6-XL5- StemPro ™/hESStemPro ™/hES hES medium Aza pre- UTF1 + medium + ActivinA medium +ActivinA treated pCMV6-XL4- (30 ng/ml) + (30 ng/ml) + DPPA4 + CHIR99021(3 μM) CHIR99021 (3 μM) pCMV6-XL5- MBD2 11 VPA + 5- pCMV6-XL4-StemPro ™/hES StemPro ™/hES hES medium Aza pre- Oct4 + medium + ActivinAmedium + ActivinA treated pCMV6-XL5- (30 ng/ml) + (30 ng/ml) + Sox2 +CHIR99021 (3 μM) + CHIR99021 (3 μM) pCMV6-XL5- VPA + 5- FoxD3 Aza 12VPA + 5- pCMV6-XL4- StemPro ™/hES StemPro ™/hES hES medium Aza pre-Oct4 + medium + ActivinA medium + ActivinA treated pCMV6-XL5- (30ng/ml) + (30 ng/ml) + Sox2 + CHIR99021 (3 μM) + CHIR99021 (3 μM)pCMV6-XL5-UTF1 VPA + 5- Aza 13 VPA + 5- pCMV6-XL4- StemPro ™/hESStemPro ™/hES hES medium Aza pre- Oct4 + medium + ActivinA medium +ActivinA treated pCMV6-XL5- (30 ng/ml) + (30 ng/ml) + Sox2 + CHIR99021(3 μM) + CHIR99021 (3 μM) pCMV6-XL4- VPA + 5- DPPA4 Aza 14 VPA + 5-pCMV6-XL4- StemPro ™/hES StemPro ™/hES hES medium Aza pre- Oct4 +medium + ActivinA medium + ActivinA treated pCMV6-XL5- (30 ng/ml) + (30ng/ml) + FoxD3 + CHIR99021 (3 μM) + CHIR99021 (3 μM) pCMV6-XL5-UTF1VPA + 5- Aza 15 VPA + 5- pCMV6-XL4- StemPro ™/hES StemPro ™/hES hESmedium Aza pre- Oct4 + medium + ActivinA medium + ActivinA treatedpCMV6-XL5- (30 ng/ml) + (30 ng/ml) + FoxD3 + CHIR99021 (3 μM) +CHIR99021 (3 μM) pCMV6-XL4- VPA + 5- DPPA4 Aza 16 VPA + 5- pCMV6-XL4-StemPro ™/hES StemPro ™/hES hES medium Aza pre- Oct4 + medium + ActivinAmedium + ActivinA treated pCMV6-XL5- (30 ng/ml) + (30 ng/ml) + UTF1 +CHIR99021 (3 μM) + CHIR99021 (3 μM) pCMV6-XL4- VPA + 5- DPPA4 Aza 17VPA + 5- pCMV6-XL5- StemPro ™/hES StemPro ™/hES hES medium Aza pre-Sox2 + medium + ActivinA medium + ActivinA treated pCMV6-XL5- (30ng/ml) + (30 ng/ml) + FoxD3 + CHIR99021 (3 μM) + CHIR99021 (3 μM)pCMV6-XL5-UTF1 VPA + 5- Aza 18 VPA + 5- pCMV6-XL5- StemPro ™/hESStemPro ™/hES hES medium Aza pre- Sox2 + medium + ActivinA medium +ActivinA treated pCMV6-XL5- (30 ng/ml) + (30 ng/ml) + FoxD3 + CHIR99021(3 μM) + CHIR99021 (3 μM) pCMV6-XL4- VPA + 5- DPPA4 Aza 19 VPA + 5-pCMV6-XL5- StemPro ™/hES StemPro ™/hES hES medium Aza pre- Sox2 +medium + ActivinA medium + ActivinA treated pCMV6-XL5- (30 ng/ml) + (30ng/ml) + UTF1 + CHIR99021 (3 μM) + CHIR99021 (3 μM) pCMV6-XL4- VPA + 5-DPPA4 Aza 20 VPA + 5- pCMV6-XL5- StemPro ™/hES StemPro ™/hES hES mediumAza pre- FoxD3 + medium + ActivinA medium + ActivinA treated pCMV6-XL5-(30 ng/ml) + (30 ng/ml) + UTF1 + CHIR99021 (3 μM) + CHIR99021 (3 μM)pCMV6-XL4- VPA + 5- DPPA4 Aza 21 VPA + 5- GFP StemPro ™/hESStemPro ™/hES hES medium Aza pre- medium + ActivinA medium + ActivinAtreated (30 ng/ml) + (30 ng/ml) + CHIR99021 (3 μM) CHIR99021 (3 μM)+/or− VPA + 5-Aza

In order to characterize subpopulations of cells after transfection,live staining, immunohistochemistry, and AP staining was performed tofollow the change in pluripotent markers. Cells transfected with eitherOct4/UTF1/MBD2, Oct4/Dppa4/MBD2, FoxD3/Dppa4/MBD2, Oct4/FoxD3/Dppa4, orSox2/FoxD3/UTF1 showed positive colonies for TRA1-60, TRA1-81, andSSEA4. This observation indicated that MBD2 generally had no effect byitself on reprogramming towards pluripotent-like cells, except in thecase of Oct4/FoxD3/MBD2 transfection. Colonies started to form on Day 7and continued to form until Day 14 (FIG. 18) (the end of the studyperiod). These colonies were positive for AP as well.

These results were confirmed by RT-PCR analysis showing up-regulation ofOct4 expression as shown in Table 35. Relative expression for SOX2 wasalso slightly up-regulation in Day 7 after transfecting cells withOct4/Foxd3/MBD2. There is also a trend of Sox2 up-regulation followingtransfection with Oct4/Sox2/Foxd3 and Oct4/Foxd3/Utf1.

TABLE 35 Relative expression of Pluripotent genes after transfectingADSCs with various combinations of vectors as described in Table 34.Endogenous OCT4 SOX2 Rel. Std. Rel. Std. Exp. Dev. Exp. Dev. #1 Day 7,Oct4/Sox2/MBD2 25.20 1.89 3.89 2.06 #2 Day 7, Oct4/Foxd3/MBD2 11.28 0.1318.79 0.03 #3 Day 7, Oct4/Utf1/MBD2 2.01 0.20 2.93 1.73 #4 Day 7,Oct4/Dppa4/MBD2 9.68 1.36 1.18 0.15 #5 Day 7, Sox2/Foxd3/MBD2 1.06 0.552.68 2.90 #6 Day 7, Sox2/Utf1/MBD2 0.66 0.10 3.36 0.68 #7 Day 7,Sox2/Dppa4/MBD2 0.74 0.00 5.03 4.73 #8 Day 7, Foxd3/Utf1/MBD2 1.31 0.614.15 2.92 #9 Day 7, Foxd3/Dppa4/MBD2 0.63 0.02 3.90 2.17 #10 Day 7,Utf1/Dppa4/MBD2 0.96 0.04 4.97 1.92 #11 Day 7, Oct4/Sox2/Foxd3 48.171.89 7.68 1.79 #12 Day 7, Oct4/Sox2/Utf1 48.97 6.93 3.71 0.39 #13 Day 7,Oct4/Sox2/Dppa4 32.40 2.74 4.61 2.37 #14 Day 7, Oct4/Foxd3/Utf1 4.300.91 9.83 3.03 #15 Day 7, Oct4/Foxd3/Dppa4 4.21 0.11 4.57 0.85 #16 Day7, Oct4/Utf1/Dppa4 10.29 3.70 3.53 1.63 #17 Day 7, Sox2/Foxd3/Utf1 1.420.83 3.32 2.12 #18 Day 7, Sox2/Foxd3/Dppa4 1.19 0.14 3.37 1.23 #19 Day7, Sox2/Utf1/Dppa4 1.34 0.09 2.33 2.91 #20 Day 7, Foxd3/Utf1/Dppa4 0.720.07 2.45 0.27 #21 Day 7, GFP (−VPA/−5aza) 1.02 0.29 1.01 0.17 #22 Day7, Untransf. ADSC (−VPA/−5aza) 1.25 N/A 0.30 N/A #23 Day 7, GFP(+VPA/+5aza) 1.01 0.20 1.87 2.23 #24 Day 7, Untransf. ADSC 1.45 N/A 0.27N/A (+VPA/+5aza)Reprogramming Efficiency of Defined Pluripotency Factors on HFF afterTriple Transfection (One Transfection Every 3 Days)

HFF cells were cultured as described in Example I with the exception ofthe concentrations of VPA and 5-AZA that were respectively 2 mM and 2.5μM. Cells were transfected using the Nucleofector™® II Device (Lonza)following procedure described in Example II with the exception of theDNA quantity: 1 μg of each of the 3 plasmids DNA was used. The cellsthat had been pre-treated with VPA and 5-Aza and the untreated cellswere both transfected with a mix of pCMV-Oct4nuc-IRES2-Sox2nuc,pCMV-Klf4nuc-IRES2-Cmycnuc or pCMV-Nanognuc-IRES2-Lin28. Followingtransfection the cells were plated in the fibroblasts medium describedin Example I, supplemented with or without VPA and 5-AZA on Matrigel™(BDBiosciences) coated on 6-well plates and incubated at 37° C., 5% CO₂.On Day 1 and 2, media was changed to 100% mTeSR1 medium (StemCellTechnologies) supplemented with or without VPA and 5-AZA. On Day 3 andDay 6, cells from each condition were detached by incubation in TrypLE™for 5 min, counted and centrifuged. Cells were retransfected as aboveand plated on Matrigel™ coated plates in mTeSR1 medium supplemented withor without VPA and 5-AZA. Media was changed daily as described for day 1and 2. Medium was supplemented in Y27632 (Stemgent, 10 μM) from day 7 today 14 to promote viability and clonal expansion of potentialreprogrammed cells. Cells were analysed at Day 20 using the AlkalinePhosphatase Detection Kit (Millipore) and by immunohistochemistryanalysis.

This analysis revealed that after three transfections, three clones werefound to be positive for alkaline phosphatase activity and showed arounded cell/colony morphology. Staining with antibodies against theembryonic stem (ES) cell markers SSEA-4 and TRA-1-81 confirmed thatthese clones were pluripotent-like (FIG. 19). Surrounding HFF cells werenegative for these markers. These clones were obtained only in thecondition that did not contain inhibitors (i.e.: VPA and 5-AZA).Unexpectedly, no clones were observed for the condition treated withthese inhibitors.

Reprogramming of NSLCs into Pluripotency

NSLC and neuronal stem cells derived from BG-01, a human ES cell linethat expresses markers that are characteristic of ES cells includingSSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-3/4, were reprogrammed intopluripotency. BG-01 cells had previously been cultured in conditions toinduce the differentiation towards neural stem cells as described byChambers S M et al., 2009. NSLCs and BG-01-NSC were cultured inproliferation medium supplemented with FGF (20 ng/ml) and EGF (20ng/ml). NSLCs and BG-01-NSCs were transfected as previously described inExample II by two episomal vectors, pEF-Oct4nuc-IRES2-MBD2 (NC1) orpCMV-FoxD3-2A-Oct4-2A-Klf4 (F72). Following transfection cells werecollected and plated onto uncoated petri-dishes in the presence ofProliferation medium and mTeSR1 medium (50:50) in proliferationconditions at 37° C., 5% CO2. After 48 hours, cells were re-transfectedby the same plasmid and plated in 96-well plates coated with Matrigel™and cultured in the presence of mTeSR1 medium supplemented by the smallmolecules BIX01294 (Stemgent, 2 μM) and BayK8644 (Stemgent, 2 μM) at 37°C., 5% O₂ for 22 days. Live staining and immunohistochemistry wereperformed to characterize subpopulations of cells for pluripotencymarkers.

NSLCs and BG-01-NSCs were positively stained with SSEA-4 starting on Day7 and maintained until 22 days in culture (the end of the study) (FIG.20). Within ten days, cells that were morphologically similar to ESCswere observed and they expressed a wide panel of pluripotency markers,including SSEA-4, TRA1-81, Nanog and Oct4 (FIG. 20). This studyidentified another way to get pluripotent-like cells from somatic cellsvia Neural Stem-Like Cells (NSLCs). The utility of NSLCs could offermultiple advantages for reprogramming towards pluripotent-like cells.For example, obviating the requirement for tumorigenic genes like c-Mycreduces the risk of inducing cancerous cells. For neuroregenerative andneurodegenerative applications these cells could represent an invaluablesource of cells to investigate furthermore human pluripotent cellinduction, and also represent a potential source of cells for derivingpatient-specific multipotent and pluripotent stem cells for modelinghuman disease.

Example XX Teratoma Formation Assay in SCID Mice

Transplantation of human pluripotent stem cells (SC) into “severelycompromised immuno-deficient” (SCID) mice leads to the formation ofdifferentiated tumors comprising all three germ layers for pluripotentstem cells, resembling spontaneous human teratomas, and specializedtissue for multipotent stem cells. These assays are considered thestandards in the literature for demonstrating differentiation potentialof pluripotent stem cells and hold promise as a standard for assessingsafety among SC-derived cell populations intended for therapeuticapplications

After all appropriate animal approvals for the experiment has beenobtained, 24 mice were purchased from Charles Rivers, and housed atMISPRO animal facility for one week without any experimentation foradaption to the new environment. One million human NSLCs, normal humanneuroprogenitor cells (hNPCs), or human embryonic stem (ES) cells in 100μl Phosphate buffered saline, calcium- and magnesium-free (CMF-PBS) wereinjected with a 21-G needle intramuscularly into the right hind limb ofthe 4-week-old male SCID-beige mice under anesthesia withKetamine/xylazine (8 mice per group). Following injection, the syringewas aspirated up and down a couple of times in a culture dish containingmedium to verify that the cells were injected and not stuck inside thesyringe.

The mice were maintained for 12 weeks and monitored for clinical signsand any tumor growth regularly. Any specialized tissue or teratomagrowth was monitored by external examination and an increase in the sizeof the muscle relative to the same muscle on the left hind limb. When aspecialized tissue or teratoma was identified, the location and size ofthe growth was measured (using measuring calipers) and recorded. Thespecialized tissue or teratoma is usually first identified as a smallgrowth of the muscle size compared to the left control muscle. Animalswere monitored weekly until onset of any tumor growth, and daily aftertumors appeared. After 12 weeks, the mice were sacrificed by CO₂euthanasia. Each entire animal was observed for any tumor growthanywhere on the animal, and the injected muscle and the comparable leftmuscle control were measured (with measuring calipers)(see results tablebelow) and then removed and stored in 4% paraformaldehyde solution forhistological analysis. The sizes of the muscles were as follows:

Left leg (control) Right leg (treated) Dorso- Dorso- ventral ventralTreatment width Lateral width width Lateral width Human 6.44 ± 0.11 5.03± 0.17 6.91 ± 0.15  5.3 ± 0.14 Embryonic Stem Cells Human 6.60 ± 0.175.43 ± 0.15 7.01 ± 0.23 5.58 ± 0.13 Neuro- progenitor Cells Human NSLC6.85 ± 0.2  5.32 ± 0.14 6.86 ± 0.21 5.33 ± 0.11 Values represent theAverage of 8 mice ± the standard error

Measurement of the size of the muscles revealed that all the humanembryonic stem cell injected muscles were bigger than the comparableleft muscle controls, indicating teratoma growth in the ES cell injectedmuscles. About half of all the human neuroprogenitor cell injectedmuscles were bigger than the comparable left muscle controls, while themice injected with NSLC did not show any difference between the muscles(treated with the cells or not). The mice injected with NSLC did notshow any evidence of tumor or teratoma growth.

Example XXI Transfection of ADSCs by Various Combinations of Genes andTreatment with Small Molecules for Reprogramming to Mesendoderm-LikeCells

Human Adipose-Derived Stem Cells (ADSCs) were purchased from Invitrogenand expanded in complete StemPro™ MSC serum-free medium (Invitrogen onCellStar™ coated flasks (diluted 1:100 in PBS containing Ca²⁺/Mg²⁺) at acell density of 1×10⁴ cells/cm². ADSCs were cultured as described incomplete StemPro medium (Invitrogen) and were pretreated for 2 daysprior to transfection with VPA (1 mM) and 5-Aza (0.5 μM). Cells weretransfected using the Nucleofector® 96-well Shuffle Device (Lonza)following the procedure described previously. The cells were transfectedwith various combinations of cDNA clones summarized in Table 36. Aftertransfection, the cells were plated on 0.1% Gelatin-coated plates andincubated at 37° C., 5% CO₂, 5% O₂. Medium was changed every other dayaccording to Table 37. Cells were analyzed at Day 10 by QuantitativeRealtime PCR.

TABLE 36 Various combinations of plasmids with potential to transfectthe cells towards mesendoderm lineage. Day −2 to Day 0 Plasmidstransfected at Day 0¹ 1 Untreated FoxD3, Ngn3, Sox17, MBD2 2 FoxD3,Eomes, Gata, Mixl1, MBD2 3 Pre-treated Eomes, FoxD3, Gata6, Mixl1 4 withEomes, FoxD3, Gata6, Sox17 5 VPA & 5-Aza Eames, Gata6, Mixl1, T 6 FoxD3,Gata4, Mixl1, Sox17 7 Gata4, Gata 6, Mixl1, Oct4 8 Gata4, Mixl1, Sox17,T 9 Eomes, Mixl1, Sox17 10 Eomes, Gata6, Sox17 11 Gata4, Mixl1, Sox17 12Gata6, Mixl1, Sox17 13 GFP ¹wthere FoxD3 = pCMV6-XL5-FoxD3, MBD2 =pCMV6-AC-MBD2, T = pCMV6-XL5-T, Mixl1 = pCMV6-XL5-Mixl1, Sox17 =pCMV6-XL4-SOX17, Gata4 = pCMV6-XL5-Gata4, Eomes = pCMV6-XL4-Eomes, andGata6 = pCMV6-XL6-Gata6, All clones were purchased from Grigene andprepared using the EndoFree Plasmid Maxi Kit (Qiagen).

TABLE 37 Medium composition from Day −2 to Day 20 Day −2 to 0 Day 1 Day2 to day 3 Day 4 to day 7 Day 8 to day 10 Day 11 to day 2 StemPro CDM II(50%) + IMDM/F12 + IMDM/F12 + IMDM/F12 + IMDM/F12 + medium + IMDM/F12NEAA + NEAA + NEAA + NEAA + VPA + 5Aza (50%) + NEAA + ITS + HSA + ITS +HSA + ITS + HAS + ITS + HSA + ITS + HSA + bFGF + EGF + bFGF + EGF +bFGF + EGF + Exendin-4, bFGF + EGF + VPA + Activin Activin A + BMP4nicotinamide VPA + Activin A + 5-Aza + 5-Aza + BMP4 A + 5-Aza BMP4²Supplements added to media at the following concentrations: Activin A(Peprotech, 30 ng/mL), HSA (Baxter, 0.5%), NEAA (Gibeo, 1X), ITS (Gibeo,1X), EGF (Peprotech, 5 ng/ml), bFGF (Peprotech, 10 ng/ml), VPA(Stemgent, 1 mM), 5-Aza (Sigma, 0.5 uM), BMP4 (Peprotech, 10 ng/ml),Exendin-4 (50 ng/ml, Cat Nb: 24463, Anaspec), and Nicotinamide (10 mMfinal, Cat Nb: N5535, Sigma).

Cells were collected on Day 10 by detaching with TrypLE, followed bycentrifugation at 80.times.g for 5 minutes. Supernatant was aspiratedand the cell pellet was frozen at −86° C. until ready for RNA Isolation.RNA isolation and quantification was performed as previously described:RNA isolation and cDNA was prepared using the High Capacity cDNA RT kit(Applied Biosystems) as per the manufacturer's instructions with a finalcDNA concentration of 2 ng/μl. Real-time PCR was then performed for eachgene of interest using the FAST PCR master mix (Applied Biosystems) andthe Taqman® Gene Expression Assays (Applied Biosystems) listed below:

Taqman ® Forward Reverse Assay Primer Primer Probe Gene ID SequenceSequence Sequence GAPDH Hs99999905_m1 (house- keeper) PPIA Hs99999904_m1(house- keeper) FOXA2 Hs00232764_m1 SOX17 Hs00751752_s1 Endoge-Hs00610073_g1 nous T GSC Hs00418279_m1 CXCR4 Hs00607978_s1 GATA4Hs00171403_m1 CER1 Hs00193796_m1 CDH1 Hs01023B94_m1 (E-cad- herin) p63Hs00978340_m1 SOX2 SOX2_1078-ANY CCACCTAC GACCACCG CTGGCATG AGCATGTCAACCCATG GCTCTTG CTACTC GA (SEQ ID (SEQ ID (SEQ ID NO: 1) NO: 2) NO: 3)

Ten days following the transfection with various combinations of Oct4,Sox17, FoxD3, T, MixI1, FoxA2, and MBD2 cDNA, analysis by RT-PCR toinvestigate the expression of Mesoendoderm and mesendodermdifferentiation genes was performed. As summarized in Table 38, theRelative Expression of Sox17 and FoxA2, the markers for Endoderm andMesoderm, was up-regulated noticeably in theFoxD3/MixI1/Ngn3/MBD2-transfected sample, while the expression ofmesendoderm differentiation (ex. pancreatic lineage) genes are lowindicating an early mesendoderm phenotype. Ten days followingtransfection, SOX17 is still highly expressed in the SOX17-transfectedsamples as compared to the untreated ADCs sample that representsleftover plasmid DNA (exogenous SOX17) that still remains 10 dayspost-transfection, and any endogenous SOX17 expression that may havebeen induced.

TABLE 38 Relative Expression of the mesendoderm genes FoxA2 and Sox17after transfecting ADSCs once with various gene combinations withpotential to reprogram cells into mesoendoderm-like cells. FoxA2 Sox17Gata4 EndoPdX1 Sox9 Ngn3 Nkx2.2 Insulin Rel. Std. Rel. Std. Rel. Std.Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Day 10 Exp. Dev. Exp.Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.Foxd3/Sox17/Ngn3/ 1.13 0.06 44.41 0.51 0.16 0.11 1.13 0.06 0.98 0.051.13 0.06 1.13 0.06 1.13 0.06 MBD2 Foxd3/Mixl1/Ngn3/ 48.03 2.28 11.170.58 0.08 0.00 1.71 0.79 0.97 0.08 1.13 0.04 1.51 0.51 1.13 0.04 MBD2Foxd3/Mixl1/Pdx1/MBD2 2.36 1.68 0.33 0.07 0.08 0.00 1.19 0.03 1.24 0.081.19 0.03 1.19 0.03 1.66 0.63 Eomes/FoxD3/Gata6/ 3.12 0.06 0.05 0.000.22 0.00 3.12 0.06 1.87 0.02 3.12 0.06 3.12 0.06 3.12 0.06 Mixl1Eomes/FoxD3/Gata6/ 2.64 0.01 137.24 12.01 0.71 0.70 2.64 0.01 1.73 0.022.64 0.01 2.64 0.01 2.64 0.01 Sox17 Eomes/Gata6/Mixl1/T 1.51 0.02 1.370.05 0.11 0.00 1.51 0.02 1.46 0.03 1.51 0.02 1.51 0.02 1.51 0.02FoxD3/Gata4/Mixl1/ 0.78 0.00 84.72 3.22 0.21 0.00 0.78 0.00 1.19 0.120.55 0.00 0.78 0.00 0.26 0.00 Sox17 Gata4/Gata6/Mixl1/ 1.35 0.04 3.201.20 0.36 0.01 1.35 0.04 1.33 0.10 0.95 0.03 1.35 0.04 0.45 0.01 oct4Gata/Mixl1/Sox17/T 1.20 0.21 228.37 5.78 1.48 0.93 0.41 0.00 0.62 0.020.29 0.00 0.41 0.00 0.14 0.00 Eomes/Mixl1/Sox17 1.72 1.41 139.00 7.360.20 0.01 0.76 0.05 1.21 0.03 0.54 0.04 0.76 0.05 0.44 0.28Eomes/Gata6/Sox17 0.57 0.03 573.14 65.66 0.15 0.01 0.93 0.49 0.97 0.050.40 0.02 1.55 1.38 0.19 0.01 Gata4/Mixl1/Sox17 1.23 0.02 253.21 45.51123.32 62.32 1.52 0.21 2.32 0.04 0.58 0.01 2.25 0.65 0.98 0.01Gata6/Mixl1/Sox17 1.20 0.32 354.21 7.65 0.32 0.01 1.52 0.52 1.23 0.050.98 0.12 2.32 0.54 0.78 0.08 ADCs untransf. 0.49 0.02 0.93 0.19 0.490.48 0.49 0.02 1.16 0.08 0.34 0.01 0.73 0.33 0.16 0.01

Example XXII Transfection of ADSCs by Various Combinations of Genes andTreatment with Small Molecules for Reprogramming to PancreaticProgenitor-Like Cells

Reprogramming ADSCs into Pancreatic Progenitor-Like Cells: Human ADSCswere purchased from Invitrogen and expanded in complete StemPro™ MSCserum-free medium (Invitrogen on CellStart™ coated flasks (diluted 1:100in PBS containing Ca²⁺/Mg²⁺) at a cell density of 1×10⁴ cells/cm². Cellswere transfected using the Nucleofector® 96-well Shuttle® Device (Lonza)following the procedure described previously. The cells were transfectedwith various combinations of cDNA clones as described in Table 4. Aftertransfection, the cells were plated on Fibronectin-coated collagen gelsand incubated at 37° C., 5% CO₂, 5% O₂. Plates were coated withfibronectin (BD Biosciences) at a concentration of 1.9 .mu.g/well. Mediawas changed every other day according to Table 39. Followingtransfection cells will be cultured using high concentration of activinA (50 ng/ml) and BMP (30 ng/ml) to push the reprogramming towards themesendoderm. To initiate the transition of definitive endoderm toPrimitive Gut tube, the medium was supplemented by FGF 10 (Peprotech)and cyclopamine (Stemgent). Thereafter, the gut tube was exposed toRetinoic acid, cyclopamine, and FGF10. In order to stimulate theexpression of NGN3 and NKx2.2, the medium was supplemented by exendin 4and hepatocyte growth factor.

TABLE 39 Plasmids and media composition from Day 0 to Day 20 MediaComposition¹ Plasmids transfected at Day −2 to Day 15 and Day 0 Day 0Day 1 to 3 Day 3 to 6 Day 7 to 9 Day 10 to 14 over 1 - MBD2/Oct4/Sox17StemPro StemPro/ RPMI, B27 DMEM free DMEM free DMEM/F12 2 -MBD2/Oct4/Pdx1 medium RPMI with 1%. With of of nicotinamide 3 -Sox17/PDX1/Ngn3 17.5 mM 1 mM glucose/F12 glucose/F12 (10 nM), + 4 -MBD2/Ngn3/Sox17 glucose, glutamine, (1:1) + B27 (1:1) + B27 Hepatocyte5 - MBD2/FoxA2/NGN3 ITS, 1 mM 1% HSA + 1%, 1% 1%, 1% growth factor 6 -MBD2/PDX1/Ngn3 glutamine, FGF 10 HSA, HSA, (20 ng/ml) & 7 - Oct4/Sox17/1% HSA + (10 ng/ml) Retinoic acid Exendin4 exendin4 Pdx1/Ngn3 Activin A(2 nM) (10 nM) & (10 nM) 8 - Oct4/Pdx1/Ngn3 (50 ng/ml) + CyclopamineCyclopamine 9 - FoxA2/Pdx1/Sox17 BMP4 (10 mM), (10 mM), 10 - GFP (30ng/ml) FGF10 (10 ng/ml) ¹Supplements added to media at the followingconcentrations: Activin A (Peprotech, 30 ng/ml), HSA (Baxter, 0.5%),NEAA (Gibco, 1X), ITS (Gibco, 1X), B27 (Gibco, 1%), bFGF (Peprotech, 10ng/ml),,, VPA (Stemgent, 1 mM), 5-Aza (Sigma, 0.5 uM), Retinoic Acid(Sigma, 2 uM), FGF10 (Peprotech, 50 ng/ml), Cyclopamine (Stemgent, 2.5uM), Exendin-4 (50 ng/ml, Cat Nb: 24463, Anaspec).

Cells were collected on Day 20 to be analyzed by Quantitative Real-timePCR and RNA isolation and quantification was performed as describedpreviously. cDNA was prepared and quantitative real-time PCR wasperformed with the following Taqman® Gene Expression Assays (AppliedBiosystems) used:

Taqman ® Forward Reverse Assay Primer Primer Probe Gene ID SequenceSequence Sequence GAPDH Hs99999905_m1 (house- keeper) PPIA Hs99999904_m1(house- keeper) FOXA2 Hs00232764_m1 SOX17 Hs00751752_s1 GATA4Hs00171403_m1 Endo- PDX1_1201 GGCCCTCT GTAGGAGG ACAGCCAC genous TTTAGTGAGCAGGGAT AAACAACG PDX1 TACTGGAT GTG T SOX9 Hs00165814_m1 NGN3Hs01875204_s1 NKX2-2 Hs00159616_m1 PAX4 Hs00173014_m1 INS Hs02741908_m1CXCR4 Hs00607978_s1

RT-PCR was used to evaluate the expression of pancreatic-related genesfrom cells after 20 days post-transfection with different combinationsof genes (Table 40). This study revealed that FoxA2 (13.12±0.06), Nkx2.2(23.12±0.06) and Gata4 (5.21±2.36), markers for Endoderm and Mesoderm,were up-regulated for the Sox17/Ngn3/Pdx1-transfected sample as comparedto the GFP mock-transfected control sample. Addition of Oct4 to thisgene combination increased Gata4 expression as well as Sox17 andendogenous Pdx1 expression, while both gene combinations expressedinsulin indicative of islet .beta.-like cells.

TABLE 40 Relative Expression of mesendoderm, pancreatic progenitor, andislet β-cell genes after transfecting ADSCs once with various genecombinations with potential to reprogram cells into pancreaticprogenitor like cells (including mesoendoderm-like cells and isletβ-like cells). FoxA2 Sox17 Gata4 EndoPdx1 Sox9 Ngn3 Nkx2.2 Insulin Rel.Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel. Std. Rel.Std. Day 20 Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.Exp. Dev. Exp. Dev. MBD2/Oct4/Sox17 1.13 0.06 62.97 27.53 0.16 0.11 1.130.06 0.98 0.05 1.13 0.06 1.13 0.06 1.13 0.06 MBD2/Oct4/Pdx1 4.72 3.3610.59 0.29 0.08 0.00 15.91 0.03 0.97 0.08 1.13 0.04 1.51 0.51 1.13 0.04MBD2/FoxA2/PDX1 43.16 6.34 0.33 0.07 0.08 0.00 14.69 2.26 1.24 0.08 1.190.03 1.19 0.03 1.66 0.63 Sox17/PDX1/Ngn3 13.12 0.06 72.37 27.67 5.212.38 32.08 14.55 1.87 0.02 97.04 18.77 23.12 0.06 6.12 0.06MBD2/Ngn3/Sox17 2.64 0.01 137.24 12.01 0.71 0.07 2.64 0.01 1.73 0.0212.58 3.08 2.64 0.01 2.64 0.01 MBD2/FoxA2/NGN3 72.59 0.02 10.22 0.140.11 0.00 1.51 0.02 1.46 0.03 20.33 5.17 1.51 0.02 1.51 0.02MBD2/PDX1/Ngn3 0.78 0.00 42.36 1.61 0.21 0.00 20.07 6.32 1.19 0.12 59.2414.07 0.78 0.00 0.26 0.00 Oct4/Sox17/Pdx1/Ngn3 11.35 0.04 139.00 7.3634.53 17.18 116.68 41.77 1.33 0.10 121.30 4.76 5.35 0.04 6.07 0.06Oct4/Pdx1/Ngn3 1.72 1.41 1.33 0.36 9.85 5.24 67.42 34.45 1.21 0.03 34.502.38 0.76 0.05 1.44 0.28 FoxA2/Pdx1/Sox17 100.57 0.03 143.29 16.42 0.150.01 13.19 4.24 0.97 0.05 0.40 0.02 1.55 1.38 0.19 0.01 ADSC untransf.0.04 0.01 0.21 0.08 0.08 0.00 1.16 0.01 1.02 0.13 1.16 0.01 1.71 0.771.16 0.01

Next immunohistochemical analysis was performed to evaluate proteinexpression in cells transfecting with the different gene combinations.On day 20, the cells were immunostained and examined using theCellomics™ ArrayScan VTI. Cells were fixed with a 4% formaldehyde/PBSsolution for 10 min at room temperature and subsequently permeabilizedfor 5 min with 0.1% Triton X-100 in 4% formaldehyde/PBS. After two briefwashes with PBS, unspecific antibody binding was blocked by a 30 minincubation with 5% normal goat serum in PBS. Then primary antibodieswere added in 5% normal goat serum/PBS as follows: Mouse anti-Gata4(1:100, BD) and mouse anti-FoxA2 (1:200, BD), a marker for endodermlayer. After a 2 h incubation the cells were washed 4 times for 5 mineach with 0.1% Tween/PBS. Appropriate fluorescence-tagged secondaryantibody was used for visualization; Goat anti-mouse 546 (1:200,invitrogen) prepared in 5% normal goat serum/PBS was used. Afterincubation for one hour, cells were washed in 0.1% Tween/PBS three timesfor 5 min each. The DNA stain Hoechst33342 (Invitrogen) was used as amarker of nuclei (dilution 1:5000 in PBS, 10 min incubation).Fluorescence images were taken with the Cellomics™ ArrayScan HCS Readermicroscopy system. To determine an estimate of the percentage of cellsadopting endodermal phenotypes, random fields were selected and for eachfield the total number of cells (as determined by counting Hoechststained nuclei) and the total number of cells positive were determined.The cells in the Sox17/Pdx1/Ngn3 and Oct4/Sox17/Pdx1/Ngn3 transfectedgroups significantly expressed both Gata4 and FoxA2: the number ofpositive cells was enhanced in the presence of Oct4 as shown in Table41.

TABLE 41 Percentage of positive cells for Gata4 and FoxA2 at day 20after transfection of ADSCs with different expression vectors. Day 20cell count Gata4 % FoxA2 % GFP 4825 0.21 0.23 untransfected 18646 0.010.03 MBD2/Oct4/Sox17 12563 0.17 0.35 MBD2/Oct4/Pdx1 13481 2.61 0.2MBD2/FoxA2/Pdx1 13418 0.17 0.16 Sox17/Pdx1/Ngn3 15306 11.15 16.9MBD2/Ngn3/Sox17 19867 1.32 0.31 MBD2/Ngn3/FoxA2 14984 0.33 0.42MBD2/Ngn3/Pdx1 18613 0.31 10.29 Oct4/Sox17/Pdx1/Ngn3 19515 21.07 30.02Oct4/Pdx1/Ngn3 6103 0.28 0.49 FoxA2/Pdx1/Sox17 10001 0.65 8.82 Aftertransfection cells were cultured in IMDM/F12 supplemented with differenttype of growth factors and small molecules (as described in Table 4).The percentage of immunopositive cells was determined by the Cellomics ™ArrayScan HCS Reader.

Transfection with Pdx1 Ngn3, Sox17 (and Oct4) increased significantlyselected pancreas-related genes (Pdx1, Ngn3, NKX2.2, Gata4, FoxA2)indicating a transition from endoderm to a pancreatic fate during thistime period. To evaluate pancreatic hormone secretion, pancreatic-likecells were cultured for 50 days, during which time the medium wasreplaced with fresh medium every two days. We then assayed thesupernatant for insulin in the conditioned medium by antigen-captureELISA kit (Abnova, KA0921) at different time points (day 25 and day 55)and compared to the release of insulin in control cultures. According tothe manufacturer's instructions, briefly, 96-well ELISA immunoplateswere coated with Anti-insulin (CatNb#) diluted 1/1000 in carbonatebuffer (pH 9.7) and incubated at 4° C. overnight. The following day, allwells were washed with TBS-Tween 0.5% before incubation withBlock/Sample buffer 1× at room temperature for one hour without shaking.After blocking, standards and samples were added to the plates andincubated and shaken (450±100 rpm) for 2 h at room temperature.Subsequently, after washing with TBS-Tween wash buffer, plates wereincubated for 2 h with Anti-Human insulin pAb (1:500 dilution in Block &Sample 1× Buffer) at 4° C. After incubation, plates were washed fivetimes with TBS-Tween 0.5% wash buffer and 100 μl of standard and samplewas added in the plates precoated with anti-insulin and incubated for 1hour at room temperature with shaking (450±100 rpm). Then, plates werewashed five times with TBS-Tween 0.5% wash buffer and 100 μl of TMB OneSolution was added to each well. Following 10 minutes incubation at roomtemperature with shaking (450±100 rpm) for the insulin plate, a bluecolor formed in the wells. After stopping the reaction by adding 100 μlof 1N hydrochloric acid, the absorbance was read at 450 nm on amicroplate reader (Synergy 4) within 30 minutes of stopping thereactions. Concentration of released insulin in the supernatants wasdetermined according to the standard curves. ELISA results revealed thatinsulin was released from cells transfected with Oct4/Sox17/Pdx1/Ngn3,and to a lesser extent in the other groups as summarized in Table 42.

TABLE 42 Quantification of insulin release by pancreaticprogenitor/β-like cells that had been cultured for 25 and 55 days aftertransfecting ADSCs. Concentration of Concentration of insulin (μIU/ml)insulin (μIU/ml) Insulin release at day 25 at day 50 MBD2/Oct4/Sox170.05 0.07 MBD2/Oct4/Pdx1 1.02 1.87 MBD2/FoxA2/PDX1 1.13 1.11Sox17/PDX1/Ngn3 2.3 4.32 MBD2/Npt3/Sox17 0.07 1.03 MBD2/FoxA2/NGN3 0.091.30 MBD2/PDX1/Ngn3 1.23 1.03 Oct4/Sox17/Pdx1/Ngn3 4.32 7.85Oct4/Pdx1/Ngn3 2.45 3.21 FoxA2/Pdx1/Sox17 1.02 1.23 ADSC untransfected0.01 0.03 Insulin release into the medium, at days 25 and 55, wasmeasured by antigen-capture ELISA (Abnova).

In addition to increase of gene expression, the transfecting cells wereable to release insulin even in low concentrations. Generatingreprogrammed .beta.-like cell lines that can locally deliver insulincould be used as a method to treat and allow functional recovery fromdiabetes.

Example XXIII Transfection of ADSCs by Various Combinations of Genes andTreatment with Different Small Molecules for Reprogramming to CardiacProgenitor Like Cells

ADSCs were cultured in StemPro™ MSC serum-free medium (Invitrogen) aspreviously described, and then transfected with different combinationsof cDNA clones as described in Table 43 using the Nucleofectora 96-wellShuttle® Device (Lonza) as described previously. After transfection, thecells were plated on Matrigel-coated plates and incubated at 37° C., 5%CO₂, 5% O₂. Medium was changed every other day according to Table 44.Cells were analyzed at Day 20 by Quantitative Real-time PCR and byImmunohistochemistry.

TABLE 43 Various combinations of plasmids with potential to transfectthe cells towards Cardiac Progenitor-like cells lineage. Day −3 to Day 0Plasmids transfeeted at Day 0¹ 1 Pre-treated T, FoxD3, Sox17, Mesp1 2with Foxd3, Sox17, Mesp1, Nkx2.5 3 VPA & 5-Aza Foxd3, Tbx5, Baf, Ntx2.54 Foxd3, T, Mesp1, Gata6 5 Sox17, Tbx5, Baf, Nkx2.5 6 Foxd3, T, Mesp1,Gata4 7 T, Tbx5, Baf, Nkx 2.5 8 Foxd3, T, Mesp1, Tbx5 9 Mesp1, Tbx5,Baf, Nkx2.5 10 Foxd3, T, Mesp1, Tbx5 11 Foxd3, Sox17, Mesp1, Gata6 12Foxd3, Sox17, Mesp1, Gata4 13 FoxD3, Sox17, Mesp1 Tbx5 GFP

TABLE 44 media composition from day −3 to day 20. Media Composition¹ Day−3 to 0 Day 1 Day 2 to Day 3 Day 4 to Day 6 Day 7 to Day 20 StemPRoIMDM/F12 IMDm/F12 + IMDM/F12 + IMDM/F12 + medium + (50%) + NEAA NEAA +ITS + NEAA + ITS + NEAA + ITS + VPA + 5-Aza (1X) + ITS + HSA + FGF8HSA + FGF8 HSA + FGF HAS (5 mg/ml) + (10 ng/ml) + (10 ng/ml) + (50ng/ml) + VRA + Activin 5 Aza + VRA + FGF (50 ng/ml) + BMP4 (50 ng/ml) A(30 ng/ml) + CHIR99021 5Aza + CHIR99021 CHIR99021 (2 nM) (2 nM) + Wnt 11(2 nM) + Wnt (50 ng/ml) + 11 (50 ng/ml) + BMP4 (50 ng/ml) BMP4 (50ng/ml) ¹Supplements added to media at the following concentrations:Activin A (Peprotech, 30 ng/ml), HSA (Baxter, 0.5%), NEAA (Gibco, 1X),ITS (Gibco, 1X), FGF8 (Peprotech, 10 ng/ml), bFGF (Peprotech, 50 ng/ml),CHIR99021 (Stemgent, 2 uM), VPA (Stemgent, 1 mM), 5-Aza (Sigma, 0.5 uM),BMP4 (Peprotech, 50 ng/ml)

Cells were collected on Day 20 by detaching with TrypLE, followed bycentrifugation at 80.times.g for 5 minutes. Supernatant was aspiratedand the cell pellet was frozen at −86° C. until ready for RNA Isolation.RNA isolation, cDNA preparation and quantitative real-time PCR wasperformed as previously described.

Gene expression analysis on day 20 following transfection revealed anincreased of several Mesoderm markers. Interestingly, three markers forcardiac progenitors cells (Nkx2.5, MyoCD, Tbx5) and three markers ofearly mesoderm lineage (Gata4, Mesp1, and Meox1) were highly increasedafter transfecting cells with brachyury (T)/Tbx5/Nkx2.2/Mesp1.Similarly, expression of Mesp1, Tbx5, Nkx2.5, MyoCd, and Meox1 wasincreased after transfecting the cells with Foxd3/Sox17/Mesp1/Tbx5;while expression of Mesp1, Gata4, Nkx2.5, MyoCd, and Meox1 was increasedafter transfecting with Foxd3/Sox17/Mesp1/Nkx2.5 andFoxd3/T/Mesp1/Gata4. As shown in Table 45, the Relative Expression ofmesoderm and cardiac progenitor cells markers are especiallyup-regulated in the combination of Mesp1/Tbx5/Nkx2.5/T,Foxd3/Sox17/Mesp1/Nkx2.5 or /Tbx5, and Foxd3/T/Mesp1/Gata4. In general,transfection of ADSCs with a combination Mesp1, FoxD3, Tbx5, Brachyury,Nkx2.5, Sox17 and/or Gata4 increased the expression of mesoderm andcardiac progenitors markers.

In addition, samples were collected at day 20 to evaluate the nature ofreprogrammed cells by analyzing the expression of mesoderm markers usingImmunohistochemistry analysis for Brachyury and Nkx2.5 according to themethods previously described. Brachyury was significantly expressed inthe Mesp1/Tbbx5/Nkx2.5/T, Foxd3/Sox17/Mesp1/Nkx2.5 or /Tbx5, andFoxd3/T/Mesp1/Gata4 groups, while all groups expressed Nkx2.5. Thesecells could represent and invaluable source of cells to investigatefurthermore human cardiac cells and also represent a potential source ofcells for deriving patient-specific multipotent stem cells for modeling,regenerating, or treating human cardiovascular diseases.

TABLE 45 Relative Expression Gata4, Mesp1, Tbx5, Nkx 2.5, MyoCD, andMeox1, after transfecting ADCs once with various gene combinations withpotential to reprogram ADSCs cells into Cardiac Progenitor-like cells.Gata4 MESP1 TBX5 NKX2.5 MYOCD MEOX1 Rel. Std. Rel. Std. Rel. Std. Rel.Std. Rel. Std. Rel. Std. Day 20 Exp. Dev. Exp. Dev. Exp. Dev. Exp. Dev.Exp. Dev. Exp. Dev. GFP-transfected ADCs 1.01 0.16 1.01 0.19 1.00 0.051.02 0.27 1.00 0.07 1.02 0.27 Foxd3/Sox17/T/Mesp1 0.02 0.01 6.70 1.181.52 0.12 0.94 0.07 1.98 0.13 1.37 0.10 Foxd3/Tbx5/Baf60c/Nkx2.5 0.080.03 1.86 0.20 1.59 0.08 24.94 2.97 2.11 0.11 1.17 0.15Sox17/Tbx5/Baf60c/Nkx2.5 0.62 0.09 3.89 0.10 1.45 0.00 41.72 2.35 0.900.02 1.19 0.02 T/Tbx5/Baf60c/Nkx2.5 0.93 0.12 7.59 0.61 1.76 0.12 27.542.36 0.94 0.05 1.23 0.19 Mesp1/Tbx5/Baf60c/Nkx2.5 1.12 0.13 31.55 0.914.26 0.07 154.76 3.50 1.54 0.06 5.96 1.60 Foxd3/Sox17/Mesp1/Gata6 0.710.02 19.59 0.76 1.77 0.15 31.38 1.23 1.44 0.09 3.30 0.68Foxd3/Sox17/Mesp1/Gata4 0.02 0.00 2.66 0.44 1.45 0.24 0.81 0.24 0.750.03 0.65 0.17 Foxd3/Sox17/Mesp1/Tbx5 1.75 3.18 138.28 2.20 15.61 0.8745.20 14.88 11.06 1.02 4.36 3.45 Foxd3/Sox17/Mesp1/Nkx2.5 17.76 4.3422.03 0.08 3.03 0.01 95.73 11.68 10.25 2.32 10.54 2.39Foxd3/T/Mesp1/Gata6 27.10 6.47 3.59 0.42 2.58 0.29 5.11 0.85 21.03 1.0212.99 1.31 Foxd3/T/Mesp1/Gata4 15.87 6.13 50.20 6.41 2.37 0.18 55.436.95 19.86 2.10 4.32 0.15 Foxd3/T/Mesp1/Tbx5 0.83 0.02 8.20 0.48 2.400.37 29.25 1.28 0.76 0.08 1.40 0.46 Foxd3/T/Mesp1/Nkx2.5 19.79 2.03111.35 15.32 2.56 0.35 32.52 12.53 0.29 0.03 24.70 3.12T/Tbx5/Nkx2.5/Mesp1 18.46 1.06 103.09 22.28 102.53 15.02 96.32 16.6015.32 5.01 15.73 2.83

Example XXIV Reprogramming Human ADSCs to Pluripotent-Like Stem Cells(PLSC)

Based on previous results in Example XIX, the highest reprogrammingefficiency was observed using pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP (NF10) andpCMV-Sall4-2A-Oct4-2A-Klf4-2A-Nanog (S71). ADSCs (InvitrogenCorporation) were cultured in cell culture flasks with completeStemPro-43 medium (Invitrogen) at 37° C., 5% CO₂ and the medium waschanged 3 times per week. After 3 days in culture cells (passage 5) weretrypsinized and counted to be transfected. Cells were transientlytransfected with one plasmid: pCMV-Sall4-2A-Oct4-2A-Klf4-2A-Nanog orRex1-EF-Oct4-2A-Klf4-2A-RFP (2 .mu.g) using nucleofector as described inExample II. Following the transfection cells were cultured in 6-wellplates in suspension with 50:50 ratio of adipocyte complete medium(StemPro-43) and embryonic stem cell medium (mTesR1). After two days inculture, cells were re-transfected with the same plasmids listed aboveand cells were plated in 96 well-plates coated with Matrigel (BDBiosciences) in the presence of mTesR complete medium supplemented withthiazovivin (0.5 μM), an ALK-5 inhibitor (SB 341542, Stemgent, 2 μM),and inhibitor of MEK (PD0325901, Stemgent, 0.5 μM). Medium was changedevery day and cells were cultured for 22 days at 37° C., 5% CO₂, 5% O₂.Alkaline Phosphatase Detection Kit (AP, Millipore) andimmunohistochemistry were performed to analyze the expression ofpluripotency markers. ALP staining was performed using AP detection kit(Millipore) according to manufacturer's instructions. Colonies emergedaround Day 15 and maintained in culture up to the end of the studyperiod (up to 2 months) with a stable morphology. Live staining showedthat these colonies express typical pluripotency markers, includingSSEA-4 and TRA1-81, TRA1-60, Oct4, Nanog, Sox2, and E-cadherin (FIG.25). Further analysis of these colonies showed that the colonies presentas well other ESC markers such as alkaline phosphatase (ALP, not shown).Supplementing the small molecules PD0325901 and SB431542 with the KSORmedium treated cultures, a 6 fold improvement in efficiency over theconventional method was obtained following the transfection of ADSCswith pCMV-pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP. This pattern did not changeup to the 2 month culture period and same positive colonies wereobserved after transfecting ADSCs with Sall4-2A-Oct4-2A-Klf4-Nanog (notshown). The observation over time showed that the phenotype of thesecolonies moves from an early SSEA+ phenotype to a lateOct4+/Sox2+/Nanog+ phenotype, which is closer to the final reprogrammedstate. Following the reprogramming of ADSCs cells to Pluripotent-likecells using the two vectors pEF-Rex 1-EF-Oct4-2A-Klf4-2A-RFP orpCMV-Sall4-2A-Oct4-2A-Klf4-2A-Nanog, a study was performed to examinethe differentiation capacity of these cells. Beginning on Day 30,reprogrammed cells were cultured in conditions in order to stimulate thedifferentiation to embryonic bodies (EBs). These differentiationconditions consisted of placing the cells on uncoated petri-dish in EBDifferentiation medium consisting of KSOR medium with FBS 5% without FGFfor 45 days. To further differentiate the embryonic bodies, Day 30 EBswere cultured in three different media in order to drive celldifferentiation to either ectoderm, mesoderm or endoderm lineages in CDMmedium consisting of DMEMIF12 supplemented with 2 mM Glutamine, 0.11 mM2-mercaptoethanol, 1 mM nonessential amino acids, and 0.5 mg/ml HAS (YaoS et al., PNAS 2006). This CDM medium was further supplemented withdifferent concentrations of cytokines to induce the differentiationtowards the three lineages. Ectoderm media consisted of N2/B27-CDM inthe presence of Noggin (100 ng/ml) and after 10 days NGF (20 ng/ml) wasadded to the medium and the cells were grown on laminin-coated plates.Endoderm media consisted of N211327 CDM with the presence of Activin A(100 ng/ml) and the cells were grown on Gelatin-coated plates. Mesodermmedia consisted of N2/B28-CDM in the presence of Activin A (50 ng/ml)and BMP-4 (50 ng/ml) and the cells were grown on Gelatin-coated plates.Cells that had originally been reprogrammed withpCMV-Sall4-2A-Oct4-2A-Klf4-2A-Nanog differentiated better than cellsoriginally reprogrammed with pEF-Rex1-EF-Oct4-2A-Klf4-2A-RFP whichdetached from the coated plates. Visual observation of thedifferentiation of the cells into the three lineages was performed byRT-PCR and Cellomics using the Ectoderm markers (Nestin, GFAP,Beta-tubulin, Sox2), Endoderm markers (GATA4, Brachyury) and Mesodermmarkers (GATA4, Brachyury, and Nkx2.5). RT-PCR analysis revealed theIdentification of endoderm, mesoderm and ectoderm markers for all 3embryonic germ layers (Table 46).

TABLE 46 RT-PCR analysis of three embryonic germ layers following thedifferentiation of PLSCs. CDH1 ASCL1 (E-cadherin) (MASH1) NOTCH1 Std.Rel. Std. TP63 Rel. Std. A - Ectoderm Rel. Exp. Dev. Exp. Dev. Rel. Exp.Std. Dev. Exp. Dev. S71-transf. ADSC 59180.71 2036.56 70.28 12.35123114.61 17452.81 5.47 0.19 NF10-transf. ADSC 77944.51 1747.33 48.203.27 166929.89 6007.52 6.47 0.12 Mel-2 Undifferentiated, P17 84021.24178.93 9.80 4.30 10.72 2.96 5.90 0.48 ADSC Untreated Ctrl 1.20 0.93 1.000.00 1.00 0.00 1.00 0.03 SOX17 CDX2 B - Endoderm Rel. Exp. Std. Dev.Rel. Exp. Std. Dev. S71-transf. ADSC 9.39 2.21 37.77 5.11 NF10-transf.ADSC 2.33 0.94 21.05 18.59 Mel-2 Undifferentiated, P17 0.77 0.00 4.345.05 ADSC Untreated Ctrl 1.00 0.00 1.00 0.00 FOXA2 GSC CXCR4 C -Mesoderm Rel. Exp. Std. Dev. Rel. Exp. Std. Dev. Rel. Exp. Std. Dev.S71-transf. ADSC 1193.70 51.50 0.85 0.11 512.33 45.62 NF10-transf. ADSC1662.76 160.28 0.57 0.01 352.65 39.26 Mel-2 Undifferentiated, P17 191.5318.61 0.01 0.01 105.05 10.62 ADSC Untreated Ctrl 1.00 0.00 1.00 0.061.19 0.91

The Expression of TP63 (Ectoderm/Surface Ectoderm) was up-regulated inboth the Day 71 S71-transfected sample (123.000-fold) and the Day 75NF10-transfected sample (167.000-fold) as compared to the ADSC untreatedcontrol. The expression of NOTCH1, a marker for the neuroectoderm, wasslightly up-regulated in the Day 71 S71-transfected sample (5-fold) andthe Day 75 NF10-transfected sample (6-fold) as compared to the ADSCuntreated control. Expression of CDH1/E-cadherin, surface ectodermmarker, was up-regulated in both the Day 71 S71-transfected sample(59.000-fold) and the Day 75 NF10-transfected sample (78.000-fold) ascompared to the ADSC untreated control. Furthermore, the expression ofASCL1/MASH1, a neural crest marker, was up-regulated in both the Day 71S71-transfected sample (70-fold) and the Day 75 NF10-transfected sample(48-fold) as compared to the ADSC untreated control.

The expression of definitive endodeiin markers were analyzed, such asSOX17, CDX2, and NKX2.1. The expression of SOX17 was up-regulated in theDay 71 S71-transfected sample (9-fold) as compared to the ADSC untreatedcontrol. CDX2 gene expression is up-regulated in both the Day 71S71-transfected sample (38-fold) and the Day 75 NFIO-transfected sample(27-fold) as compared to the ADSC untreated control. However, NKX2-1(Endoderm-Primitive Gut Derivative) was not expressed in any of the 4test samples at the time points tested.

Markers of Mesoderm were analyzed as well: the expression of T(Mesoderm) was up-regulated in the Day 71 S71-transfected sample(16-fold) as compared to the ADSC untreated control. However theexpression of MEOX1 OSR1 (intermediate mesoderm), and FOXF1 (lateralmesoderm) was decreased in both the S71-transfected and theNF10-transfected samples as compared to the ADSC Untreated Ctrl. Theexpression of GATA4 is up-regulated in both the Day 71 S71-transfectedsample (150-fold) and the Day 75 NF10-transfected sample (110-fold) ascompared to the ADSC untreated control.

The expression of FOXA2 (Mesendoderm, Mesoderm, Endoderm) wasup-regulated in both the Day 71 S71-transfected sample (1.100-fold) andthe Day 75 NF10-transfected sample (1.600-fold) as compared to the ADSCuntreated control. No significant change in GSC (mesoendoderm, Endoderm)expression for both the S71-transfected and the NFIO-transfected samplesas compared to the ADSC Untreated Ctrl. The expression of CXCR4, amarker for Mesoderm/Endoderm, was up-regulated in both the Day 71S71-transfected sample (500-fold) and the Day 75 NF10-transfected sample(350-fold) as compared to the ADSC untreated control.

Using immunohistochemistry, the potential of PLSCs to express anddifferentiate into the three embryonic layers markers was analyzed.Consistent with their hES-like morphology, PLSCs were able todifferentiation towards the three lineages:

Ectodermal differentiation: Nestin is strongly and widely expressedthroughout the experiment. The neural markers GFAP and BIII-tubulin arealso expressed, while Sox2 as a neural precursor marker was notdetectable at the time points tested, indicating that cells had alreadydifferentiated into the neural pathway. At day 12 and 18, many cellsco-expressed GFAP and 131H-tubulin. At day 28, however, GFAP expressingcells have almost completely disappeared, while BIII-tubulin positivecells with well formed neurites become detectable (FIG. 26). The mediumsupported neural differentiation, but less astrocyte differentiation,since, especially at day 12 and 18, there are high amounts ofGFAP-positive collapsed cells detectable, indicating massive cell deathof this type of cells. The omnipresence of the neural markers suggestthat differentiation occurs along the neural pathway in this media;although different differentiation lineages could be achieved withdifferent media compositions and growth substrates/growth conditionsknown in the art. By Day 12 of differentiation, the expression of thestem cell markers Oct4 and Nanog are completely suppressed.

Endodermal differentiation: Large fields of cells that are positive forFoxA2 are detectable indicating endodermal differentiation.

Mesodermal differentiation: Cells treated for mesodermal differentiationcease to express Oct4 and nanog (not shown), indicating that they indeedstarted to differentiate. The strong and ubiquitous expression of GATA4and the later appearance of nuclear Nkx2.5 stain indicate that manycells might develop a cardiac profile in the conditions tested.

There are some cell clusters that express brachyury staining togetherwith GATA4.

As for the Ectoderm and Endoderm differentiation, different mediacompositions and growth substrates/growth conditions known in the artwould promote the acquisition of other differentiation lineages.

Example XXV Cytotoxic Effects of Human PLSCs

Human Hepatocellular Carcinoma cell line (HepG2, CRL-10741, ATCC) wereseeded in a 24 well plate (1×10⁴ cells/cm²) in DMEM with 10% Fetalbovine serum (FBS). PLSCs were harvested using dispase and seeded (1×10⁴cells/cm²) in transwells (Corning, 0.4 μm) above the HepG2 containingwells. The HepG2 cells were analyzed for cytotoxicity after 24 hours byhigh-content analysis staining by Propodium Iodide, Yoprol, and EthidiumHeterodimer (cell-impermeable DNA stains that stain only cells with acompromised plasma membrane as occurs in unhealthy cells) along withCalcein (a live stain, marking enzymatic activity in the cytoplasm),Mitotracker Red (marks active, healthy mitochondria) and Cleaved Caspase3 (a marker for apoptotic or stressed cells). HepG2 cells in thepresence of PLSCs had significantly less Propodium Iodide, Yoprol andCleaved Caspase 3 staining indicating that the PLSCs had a protectiveand/or therapeutic/regenerative effect on these cells (Table 47).

TABLE 47 The percentage of Yopro1, Propidium, ethidium Heterodimer,Calcein AM, Mitotracker Red, and Cleaved Caspase-3 positive cells foruntreated and PLSCs treated hepatocytes. Average % positive cellsintensity Number of Propidium Ethidium Cleaved Mitotracker cells Yopro1Iodide Heterodimer CalceinAM Caspase3 Red HepG2 14539 ± 1359 5.15 ± 0.474.57 ± 0.21 5.66 ± 0.02 69.41 ± 1.44 0.95 ± 0.05 26.88 ± 3.52 untreatedHepG2, 12498 ± 1454 3.41 ± 0.37 3.85 ± 0.36 5.81 ± 0.33 71.67 ± 1.550.63 ± 0.03 27.99 ± 3.74 PSLC treated

Example XXVI Teratoma Formation Assay in Immunodeficient NOD-SCID Mice

Fifty million human NSLCs derived from fibroblast cells (HFF) and fiftymillions NSLCs derived from blood cells (CD34+) (according to ExamplesIV and IX), as well as twenty-five million Human embryonic stem cells(Mel2) were prepared as units of five million cells each embedded with30% Matrigel (Invitrogen) in 200 μl phosphate buffered saline which werethen injected subcutaneously into the back of individual NOD/SCID miceusing a 21-G needle within 20 minutes of preparation. Each cell line hadbeen in culture for approximately 1 month prior to the study. Thus 10mice received an injection of 5 million NSLCs derived from HFF, 10 micereceived an injection of 5 million NSLCs derived from OD34+ cells, and 5mice received an injection of 5 million Mel2 cells. All standard andappropriate animal approval and ethical committee approvals wereobtained prior to the commencement of the study.

Although the study was for 3 months, within 1 month of injection one ofthe mice injected with the Mel2 cells had to be sacrificed due to thesize of the teratoma that had formed according to the animal approvalprotocol. Less than 3 weeks later, the rest of the Mel2 injected micehad to also be sacrificed due to the size of the teratoma that hadformed. None of the 10 mice injected with NSLCs derived from fibroblastcells or the 10 mice injected with NSLCs derived from OD34+ blood cellsformed any tumors or teratomas, indicating that these NSLCs are safemultipotent stem cells.

Example XXVII Differentiation of NSLC Derived from Mia, Cd34+ Cells, andKeratinocytes to Different Neuronal Lineages

To investigate the differentiation potential of NSLCs to different typesof neurons, NSLC Neurospheres were dissociated and plated ontolaminin/poly-D-Lysine (10 μg/ml; Sigma) coated plates in NeuroCultdifferentiation medium (NeuroCult Differentiation basal, StemCellTechnologies) supplemented with NeuroCult® SM1 Neuronal Supplement andBDNF (20 ng/ml, Peprotech), bFGF (40 ng/ml, Peprotech), FGF 8 (20 ng/ml,Peprotech) and SHH (20 ng/ml, Peprotech) for 30 days. NeuroCult® SM1(StemCell Technologies) is a standardized serum-free supplementcontaining antioxidants and retinoic acid; its formulation was developedbased on the published supplement formulation identified as B27 but hasbeen optimized to give reproducibly high numbers of functional neuronswith minimal glial cell contamination (<1% GFAP+). After one month inculture, the cells were stained with the neuronal marker tyrosinehydroxylase, acetycholine, GABA, and Dopamine. All cell lines stainedpositive for each of these markers. Immunohistochemistry analysis showedthat differentiation medium supplemented with Neurocult SM1 andneurotrophic factors promoted the differentiation of NSLCs derived fromHFF, CD34⁺ cells, and Keratinocytes to dopaminergic, adrenergic, andGabanergic neurons.

This study showed that NSLCs derived from fibroblast, keratinocyte, orblood cells are capable to differentiate towards different types ofneurons in the appropriate differentiation conditions. The specificneurons could be used to treat diseases where such neurons are affectedor have been lost, for example, dopaminergic neurons in Parkinson'sdisease. Other types of multipotent stem cells and progenitor cellsprepared according to the methods in the previous examples would beexpected to reprogram or differentiate more along certain pathways withthe appropriate media and supplements, growth substrate and growthconditions known in the art.

Example XXVIII Comparison of Expression of Specific Genes in Human HFFs,NSLCs Created from the HFFs, and Human Primary Neuroprogenitor Cells

The expression of selected genes and proteins in NSLCs created from HFFsaccording to Example IV were determined. Total RNA was extracted fromcells, using Trizol following manufacturer's recommendation. Briefly,cells were examined for the expression of different genes associatedwith pluripotency: SOX2 (460 bp), OCT4a (172 bp), OCT4b (169 bp) andNANOG (276 bp); early neural markers: SOX2 (460 bp), NES (327 bp), CD133(200 bp), PAX6 (431 bp) and ASCL (220 bp); Notch signaling: NOTCH1 (126bp), NOTCH2 (475 bp), HES1 (314 bp) and HESS (265 bp); neurotrophicfactors: GDNF (389 bp) and BDNF (743 bp) and astrocyte marker GFAP (650bp) and markers of neurons: NFL (284 bp), NFM (333 bp), NFH (316 bp),SYN (289 bp), NSE (292 bp) and MAP2 (321 bp), Positive controls usedwere NT2 cells (human NT2/D1 teratomacarcinoma cell line (ATCC),NT2-derived neurons (NT2-N) and astrocytes (NT2-A) and SH-SY5Y cellswere appropriate. No template control was used as negative control forevery gene examined.

As seen in FIG. 27, NSLCs had the same expression profile as humanprimary neuroprogenitor cells (NT; Lonza), namely in terms of expressionof Sox2, Oct4b, Notch1, Notch2, Hes1, Hes5, Nestin, CD133, Pax6, AscI1(Mash1), NFL, NFM, NSE and Map2, but additionally the of theneurotrophic factors GDNF and BDNF. The HFF cells that the NSLCs werecreated from also expressed Oct4b (Oct4b is a spicing variant of Oct4a(a stem cell marker) which expression is not limited to stem cells),Nestin, some of the notch signaling genes and neuronal markers, makingit significantly easier to reprogram than adult dermal fibroblasts thatdo not express this genes (thus, for example, Nestin is important to addto the reprogramming cocktail when reprogramming adult fibroblasts toNSLCs).

Next immunohistochemistry was used to confirm that the encoded proteinof some of the above expressed genes was present in the cells. Cellswere fixed with 65% Ethanol and 0.15M NaCl for 20 minutes and stainedfor the following proteins: SOX2, NES, GFAP, MAP2, NCAM and RBPJ(green). Phase and Hoechst (blue) counter stain was also preformed. NT2and NP cells were used as comparison and positive controls. NSLCsexpressed intense staining for Sox2, Nestin, GFAP, Map2, and RBPJ withonly a small amount of NCAM detected. NP cells had an identical stainingpattern to the NSLCs except slightly less intense staining especiallyfor Sox2 and Nestin. The NT2 cells only expressed Sox2, Nestin and RBPJat appreciable levels. The HFFs from which the NSLCs were created fromonly expressed Nestin and RBPJ at appreciable levels.

Example XXIX Potential of NSLCs for CNS Therapeutic Applications

NSLCs prepared from HFFs according to Example IV were tested for theirability to form functional cell-cell communication through gapjunctions, which is an important characteristic for therapeuticpotential when implanted into the CNS. Dye coupling experiments showedfunctional cell-cell communication in NSLC cells through gap junctions.A single NSCL cell was pre-loaded with two dyes [Dil (red) and calceinAM (green)] and plated on an unlabeled layer of NSCL cells. Calceinreadily transferred from the donor cells to a large population ofreceiving cells after 3 hours in vitro, confirming gap junctionalintercellular communication among NSCL cells (FIG. 28).

Next NSLCs were tested for the expression of synaptotagmin (a synapticvesicle protein) and MAP2 (a neural differentiation associated marker)since NSLCs have some unique characteristics over native neuralstem/progenitor cells including expression of some growth factors aswell as a more significant expression of some neural differentiationmarkers while maintaining the ability to remain in a stem cell likestate. NSLCs were found to readily express synaptotagmin and Map2 (FIG.29).

Next NSLCs were tested for their tolerance to glutamate and NMDA todetermine their potential robustness in areas of significant ischemia,trauma, and neurodegeneration in the CNS. NSLCs maintained a healthyneuronal state in the presence of glutamate (200 mM), a characteristicnot observed in the presence of NMDA (25 mM), indicating an ability toform synapses upon neuronal differentiation.

Next the NSLCs were tested for their ability to attach and survive on 3Dscaffolds; in this case PGA scaffolds (FIG. 30). NSLCs were found toreadily attach to the PGA scaffolds designed for implantation into thebrain. Carboxyfluourodiacetate (CFDA) confirms optimal cell survival(>93%) 24 hours after cells were plated on the scaffold. NSLCs showedrobust attachment and survival to the surface for at least one week invitro. Furthermore, Neurites grew along the length of each PGA fiber,following the pattern of the scaffold, indicating that NSLCs can begrown on 3D scaffold and structures useful for implantation into humansor animals, or for in vitro modeling.

PATENT LITERATURE CITED

-   Bhasin, Vishal. 2010. Method of Producing Progenitor Cells from    Differentiated Cells. PCT/AU2010000120.-   Cifarelli R A, Cellini F, Liddo R D, Parnigotto et al., 2010. Method    For the Production of Multicomponent Stem Cells, Relative kits and    Used in the Medical Field. US 2010/0003223A1.-   Kremer B K F, Fandrich F, 2006. Dedifferentiated, Programmable Stem    Cells of Monocytic Origin, and Their Production and Use. U.S. Pat.    No. 7,138,257 B2.-   Winner G F. Newson B S, Rill D R, and Williams Jim C. 2010.    Monocyte-Derived Stem Cells. US2010/0047908A1.-   Bennett D et al. 2009 Stem-Like Cells and Method for Reprogramming    Adult Mammalian Somatic Cells. Wo/2009/079007.-   Akamatsu W, Okano H. Method for Producing Neural Stem Cells.    Wo/2010/052904.-   You, S, Moon J H, Yoon B S, Kim K D, Park G, Jun E K, Kim B, Yoo, S    J, Kwak S S, Maeng I. 2007. Dedifferentiation d'Astrocytes en    Cellules Souches Neuronales au Moyen du Gene BMI-1.    (WO/2007/097494).-   You, S, Moon J H, Yoon B S, Kim D, Park G, Jun E K, Kim B, Yoo, S J,    Kwak S S, Maeng I. 2009. De-Differentiation of Astrocytes into    Neural Stem Cell using Nanog. US2009/0246870A1.-   Chen S, Ding S, Schltz P G. Compositions and Methods for Inducing    Cell Dedifferentiation. US2005/0176707A1.-   You S, et al., 2009. De-Differentiation of Astrocytes into Neural    Stem Cells using SHH. Us 2009/0227023.-   Oliveri, R and Andersen, C Y. 2009. Method for Increasing the    Plasticity Level of a Cell. WO/2009/018832.-   Imai T, Tokunaga A, Yoshida T, Mikoshiba K, Nakafuku M, Okana H. Num    Protein. 2004. Expression Inhibitor making Use Musashi. Us    2004/0054140A1.-   Okano, H. 2003. RNA-Binding Protein Musahi 2. WO03/016531.

SCIENTIFIC PUBLICATION LITERATURE CITED

-   Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoka K,    Yamanaka S. Induction of pluripotent stem cells from adult human    fibroblasts by defined factors. Cell. 2007 Nov. 30; 131(5):861-72.-   Okita K, Hong H, Takahashi K, Yamanaka S. Generation of    mouse-induced pluripotent stem cells with plasmid vectors. Nat.    Protoc. 2010; 5(3):418-28.-   Yu J, Hu K, Smuga-Otto, Tian S, Stewart R, Slukvin I I, Thomson J A.    Human induced pluripotent stem cells free of vector and transgene    sequences. Science 2009 8; 324(5928):797-801.-   Zeitlow R, Lane E L, Dunnet S B, Rosser A E. Human stem cells for    CNSrepair. Cell Tissue Res. 2008; 331(1):301-22.-   Singec I, Jandial R, Crain A, Nikkhah G, and Snyde E. Y. The Leading    Edge of Stem Cell Therapeutics. Annual Review of Medicine 2007:58;    313-328.-   Mimeault, M., Hauke, R. & Batra, S. K. 2007. Stem cells: a    revolution in therapeutics-recent advances in stem cell biology and    their therapeutic applications in regenerative medicine and cancer    therapies. Clin Pharmacol Ther, 82, 252-64.-   Levesque, M F and Neuman T. Transdiffentiation of transfected    epidermal basal cells into neural progenitor cells, neuronal cells    and/or glial cells. Patent, filling date 2000.-   Shea T B. Neuritogenesis in mouse NB2a/d1 neuroblastoma cells:    triggering by calcium influx and involvement of actin and tubulin    dynamics. Cell Biol Int Rep. 1990; 14(11):967-79.-   Yeomans N D, Trier J S, Moxey P C, and Markezin E T. Maturation and    differentiation of cultured fetal stomach. Effects of    corticosteroids, pentagastrin, and cytochalasin B. Gasteroenterology    1976; 71(5):770-7.-   Paterson F C, Rudland P S. Microtubule-disrupting drugs increase the    frequency of conversion of a rat mammary epithelial stem cell line    to elongated, myoepithelial-like cells in culture. J Cell Phsiol.    1985; 125(1):135-50.-   Bouwens L. Transdifferentiation versus stem cell hypothesis for the    regeneration of islet beta-cells in the pancreas. Micro Res Tech.    1998; 43(4):332-6.-   Bouwens L. Cytokeratins and cell differentiation in the pancreas. J    Pathol. 1998b; 184(3):234-9.-   Theise N D, Nimmakayalu M, Gardner R, Illei P B, Morgan G, Teperman    L, Henegariu O, Krause D S. Liver from bone marrow in humans.    Hepatology 2000; 32(1):11-6.-   Woodbury D, Schwarz E J, Prockop D J, Black I B. Adult rat and human    bone marrow stromal cells differentiate into neurons. J Neurosci    Res. 2000; 61(4):364-70.-   Brunet, J F; Ghysen, A. Deconstructing cell determination: proneural    genes and neuronal identity. Bioessays. 1999; 21:313-318.-   Bertrand N, Castro D S, and Guillemot F. Proneural genes and the    specification of neural cell types. Nat Rev Neurosci. 2002;    3(7):517-30.-   McCormick M B, Tamimi R M, Snider L, Asakura A, Bergstrom D,    Tapscott S J. NeuroD2 and neuroD3: distinct expression patterns and    transcriptional activation potentials within the neuroD gene family.    Mol Cell Biol. 1996; 16(10):5792-800.-   Guillemot F, Lo L C, Johnson J E, Auerbach A, Anderson D J, Joyner    A I. Mammalian achaete-scute homolog 1 is required for the early    development of olfactory and autonomic neurons. Cell 1993;    75(3):463-76.-   Fode C, Gradwohl G, Morin X, Dierich A, LeMeur M, Goridis C,    Guillemot F. The bHLH protein NEUROGENIN 2 is a determination factor    for epibranchial placode-derived sensory neurons. Neuron 1998;    20(3):483-94.-   Fernandes K J L, McKenzie I A, Mill P, Smith K M, Akhavan M,    Barnabé-Heider F, Biernaskie J, Junek A, et al. A dermal niche for    multipotent adult skin-derived precursor cells. Nature Cell Biology    2004; 6:1082-1093.-   Jacobsen F, Hirsch T, Mittler D, Schulte M, Lehnhardt M, Druecke D,    Homann H H, Steinau H U, Steinstraesser L. Polybrene improves    transfection efficacy of recombinant replication-deficient    adenovirus in cutaneous cells and burned skin. J Gene Med. 2006;    8(2):138-46.-   Kearns C M, Gash D M. GDNF protects nigral dopamine neurons against    6-hydroxydopamine in vivo. Brain Res. 1995; 672(1-2):104-11.-   Gash D M, Zhang Z, Ovadia A, Cass W A, Yi A, Simmerman L, Russel D,    Martin D, Lapchak P A, Collins F, Hoffer B J, Gerhardt G A.    Functional recovery in parkinsonian monkeys treated with GDNF.    Nature 1996; 380(6571):252-5.-   Lindner M D, Winn S R, Baetge E E, Hammang J P, Gentile F T, Doherty    E, McDermott P E, Frydel B, Ullman M D, Schallert T et al.    Implantation of encapsulated catecholamine and GDNF-producing cells    in rats with unilateral dopamine depletions and parkinsonian    symptoms. Exp Neurol. 1995; 132(1):62-76.-   Kordower J H, Emborg M E, Bloch J, Ma S Y, Chu Y, Leventhal L,    McBride J, Chen E Y, Palfi S, Roitberg B Z, Brown W D, Holden J E,    et al. Neurodegeneration prevented by lentiviral vector delivery of    GDNF in primate models of Parkinson's disease. Science 2000;    290(5492):767-73.-   Martinez-Serrano A, Bjorklund A. Immortalized neural progenitor    cells for CNS gene transfer and repair. Trends Neurosci. 1997;    20(11):530-8.-   Chambers S M, Fasano C A, Papapetrou E P, Tomishima M, Sadelain M,    Studer L. Highly efficient neural conversion of human ES and iPS    cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;    27(3):275-80.

Headings are included herein for reference and to aid in locatingcertain sections These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specificationThus, the present invention is not intended to be limited to theembodiments shown herein but is to be accorded the widest scopeconsistent with the principles and novel features disclosed herein.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the present invention and scope of the appendedclaims.

What is claimed is:
 1. An in vitro human cardiac multipotent orunipotent cell possessing all of the following characteristics: i) hasthe ability to proliferate for more than 30 population doublings, andhas an average population doubling time of less than 3 days; ii) is nota cancerous cell; iii) is stable and not artificially maintained byforced gene expression and may be maintained in standard cardiac stemcell media; iv) can differentiate to a progenitor, precursor, or somaticcell; v) has the characteristics of a cardiac stem cell, cardiacprecursor cell, or cardiac progenitor cell; vi) does not exhibituncontrolled growth, teratoma formation, or tumor formation in vivo;vii) expresses one or more markers of a multipotent, unipotent orsomatic cell not characteristic of a cardiac stem cell, cardiacprecursor cell, or cardiac progenitor cell; viii) is capable ofmaintaining telomerase activity through to at least about 30 populationdoublings; ix) is derived from the reprogramming of a somatic cell, aprogenitor cell or a stem cell that exhibits at least a transientincrease in intracellular levels of at least one reprogramming agent;and x) wherein the cell comprises at least one polypeptide or anexpression vector encoding at least one polypeptide selected from thegroup consisting of: Mesp1; Nkx2.5; Tbx5; Mesp1 and Brachyury (T);Nkx2.5 and Brachyury (T); Tbx5 and Brachyury (T); Mesp1 and Nkx2.5;Mesp1 and Tbx5; Mesp1, Brachyury (T) and Nkx2.5; Mesp1, Brachyury (T)and Tbx5; Mesp1, Nkx2.5 and Tbx5; and Mesp1, Brachyury (T), Nkx2.5 andTbx5; wherein the expression vector is transiently expressed.
 2. An invitro human cardiac multipotent or unipotent cell, wherein the cellpossesses all of the following characteristics: i) expression of one ormore cardiac progenitor cell markers selected from the group consistingof Mesp1, Nkx2.5, Tbx5, MyoCD, and Gata4; ii) is capable of beingcultured in suspension or as an adherent culture; iii) is capable ofproliferating without the presence of an exogenous reprogramming agentfor over 1 month; iv) expression of one or more markers selected fromthe group consisting of Nkx2.5, Tbx5, MyoD, and Gata4 upon cardiacdifferentiation; v) expression of one or more markers of a multipotent,unipotent or somatic cell not characteristic of a cardiac stem cell,cardiac precursor cell, or cardiac progenitor cell; vi) capable ofmaintaining telomerase activity through to at least about 30 populationdoublings; vii) has the ability to proliferate for more than 30population doublings, with an average population doubling time of lessthan 3 days; and viii) wherein the cell comprises at least onepolypeptide or an expression vector encoding at least one polypeptideselected from the group consisting of: Mesp1; Nkx2.5; Tbx5; Mesp1 andBrachyury (T); Nkx2.5 and Brachyury (T); Tbx5 and Brachyury (T); Mesp1and Nkx2.5; Mesp1 and Tbx5; Mesp1, Brachyury (T) and Nkx2.5; Mesp1,Brachyury (T) and Tbx5; Mesp1, Nkx2.5 and Tbx5; and Mesp1, Brachyury(T), Nkx2.5 and Tbx5; wherein the expression vector is transientlyexpressed; and possesses one or more of the following characteristics:ix) is capable of dividing every 48 hours at low passage; x) is capableof differentiation into one or more of a cardiac progenitor cell, acardiac precursor cell, a cardiac muscle cell, or to another moredifferentiated cell type of the cardiac lineage; xi) has decreasedexpression of telomerase and one or more cardiac stem cell markers upondifferentiation; xii) has one or more morphological characteristic of acardiac stem cell or cardiac progenitor cell; xiii) is negative in atumor colony forming assay; xiv) is negative for tumor growth inSeverely Compromised Immuno-Deficient (SCID) mice; xv) is negative forteratoma growth in SCID mice; xvi) is capable of significantly improvingone or more functional measures after placement of an adequate number ofthe cells into a cardiac infarct mouse model; xvii) is capable ofsignificantly improving or maintaining one or more cardiac functionalmeasures after injecting an adequate number of the cells into a mousemodel for cardiovascular disease; xviii) is derived from thereprogramming of a somatic cell, a progenitor cell or a stem cell thatexhibits at least a transient increase in intracellular levels of atleast one reprogramming agent.
 3. An in vitro human cardiac somatic cellthat possesses all of the following characteristics: i) expresses atleast one cardiac marker selected from the group consisting of Nkx2.5,Tbx5, MyoD, and Gata4; ii) is not a cancerous cell; iii) is stable andnot artificially maintained by forced gene expression and may bemaintained in standard cardiac cell media; iv) does not exhibituncontrolled growth, teratoma formation, or tumor formation in vivo; v)expresses one or more markers of a multipotent, unipotent or somaticcell not characteristic of a cardiac cell; vi) wherein the cell isderived from the reprogramming of a somatic cell, a progenitor cell or astem cell that exhibits at least a transient increase in intracellularlevels of at least one reprogramming agent; and vii) wherein the cellcomprises at least one polypeptide or an expression vector encoding atleast one polypeptide selected from the group consisting of: Mesp1;Nkx2.5; Tbx5; Mesp1 and Brachyury (T); Nkx2.5 and Brachyury (T); Tbx5and Brachyury (T); Mesp1 and Nkx2.5; Mesp1 and Tbx5; Mesp1, Brachyury(T) and Nkx2.5; Mesp1, Brachyury (T) and Tbx5; Mesp1, Nkx2.5 and Tbx5;and Mesp1, Brachyury (T), Nkx2.5 and Tbx5; wherein the expression vectoris transiently expressed.
 4. A plurality of isolated cells of claim 1wherein the cells are organized within a three dimensional structure. 5.A plurality of isolated cells of claim 2 wherein the cells are organizedwithin a three dimensional structure.
 6. A plurality of isolated cellsof claim 3 wherein the cells are organized within a three dimensionalstructure.
 7. A pharmaceutically-acceptable composition comprising cellsaccording to claim 1 for transplantation into a patient in need thereof.8. A pharmaceutically-acceptable composition comprising cells accordingto claim 2 for transplantation into a patient in need thereof.
 9. Apharmaceutically-acceptable composition comprising cells according toclaim 3 for transplantation into a patient in need thereof.